G-beta-gamma regulated phosphatidylinositol-3&#39; kinase

ABSTRACT

The present invention relates to the discovery, identification and characterization of nucleotides that encode the G protein regulated phosphatidylinositol-3&#39; kinase, a heterodimeric enzyme which produces the intracellular messenger phosphatidylinositol (3,4,5)-triphosphate in response to activation of trimeric G protein-linked receptors. This novel protein, comprised of a catalytic subunit, p120, and a regulatory subunit, p101, is found in cells of hematopoietic origin and is involved in immune system responses which cause inflammation. The presence of p101 subunit is largely responsible for the dramatic stimulation of kinase activity in the presence of activated trimeric G proteins. The invention encompasses p101 and p120 nucleotides, host cell expression systems, p101 and p120 proteins, fusion proteins, polypeptides and peptides, antibodies to these proteins, transgenic animals that express a p101 or p120 transgene, or recombinant knock-out cells and animals that do not express the p101 or p120 gene, antagonists and agonists of the enzyme, and other compounds that modulate p101 or p120 gene expression or enzyme activity that can be used for diagnosis, drug screening, clinical trial monitoring, and/or the treatment of inflammatory disorders.

This application is a continuation in part of Ser. No. 672,211 filedJun. 27, 1996.

TABLE OF CONTENTS G-beta-gamma Regulated Phosphatidylinositol-3' Kinase

I. INTRODUCTION

II. BACKGROUND OF THE INVENTION

III. SUMMARY OF THE INVENTION

A. DEFINITIONS

IV. DESCRIPTION OF THE FIGURES

V. DETAILED DESCRIPTION OF THE INVENTION

A. THE p101 AND p120 GENES

B. p101 AND p120 PROTEINS AND POLYPEPTIDES

C. ANTIBODIES TO p101 AND p120 PROTEINS

D. DIAGNOSIS OF HEMATOPOIETIC CELL ACTIVATION DISORDERS

1. DETECTION OF THE p101 GENE AND TRANSCRIPTS

2. DETECTION OF THE p101 GENE PRODUCTS

E. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE G-PROTEIN ACTIVATED PI3KEXPRESSION OR ACTIVITY

1. In vitro SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO p101 REGULATORYSUBUNIT

2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE p101 OR p120PROTEINS

3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH p101 REGULATORYSUBUNIT/INTRACELLULAR MACROMOLECULE INTERACTION

4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE INFLAMMATORYDISORDERS

F. THE TREATMENT OF DISORDERS ASSOCIATED WITH STIMULATION OF G-PROTEINACTIVATED PI3K, INCLUDING INFLAMMATORY DISORDERS

1. INHIBITION OF p101 ADAPTOR EXPRESSION OR p101 ADAPTOR ACTIVITY TOREDUCE G PROTEIN ACTIVATED PI3K ACTIVITY AND REDUCE INFLAMMATION

2. RESTORATION OR INCREASE IN p101 REGULATORY SUBUNIT EXPRESSION ORACTIVITY TO PROMOTE IMMUNE SYSTEM ACTIVATION

G. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

1. EFFECTIVE DOSE

2. FORMULATIONS AND USE

VI. EXAMPLE: Purification and characterization of porcine Gβγ-activatedPI3K activities

A. MATERIALS & METHODS

1. PI3K ASSAYS

B. RESULTS

VIII. EXAMPLE: PEPTIDE SEQUENCING OF PORCINE GBγ-ACTIVATED

A. PI3K A AND B

B. MATERIALS AND METHODS

C. RESULTS

IX. EXAMPLE: Cloning of the cDNAs encoding porcine p120 and p101

A. MATERIALS AND METHODS

B. RESULTS

X. EXAMPLE: Cloning of the cDNAs encoding human p120 and p101

A. Human p101

B. Human p120

XI. EXAMPLE: Expression of porcine p120 and p101 in insect cells

A. MATERIALS AND METHODS

1. CONSTRUCTION OF EXPRESSION VECTORS

2. Sf9 cell transfections and production of recombinant proteins

3. Purification of Sf9-derived proteins

B. RESULTS

XII. EXAMPLE: Expression of porcine p101 and p120 in mammalian cells

A. MATERIALS AND METHODS

1. CELL CULTURE

2. CONSTRUCTION OF EXPRESSION VECTORS

3. U937 TRANSFECTION PROTOCOLS

4. COS-7 CELL TRANSFECTIONS

B. RESULTS

XIII. DEPOSIT OF CLONES

ABSTRACT

G-beta-gamma Regulated Phosphatidylinositol-3' Kinase I. INTRODUCTION

The present invention relates to novel G-protein regulatedphosphoinositide 3OH-kinase enzymes isolated from cells of hematopoieticlineage which are involved in cellular signal transduction pathways, andto the use of these novel enzymes in the treatment and diagnosis ofdisease.

II. BACKGROUND OF THE INVENTION

Phosphoinositide 3OH-kinases (PI3Ks) are a large family of enzymescapable of 3-phosphorylating at least one of the cellularphosphoinositides (Whitman et al., 1988, Nature 332:644-646; Auger etal., 1989, Cell 57:167-175). 3-phosphorylated phosphoinositides arefound in all higher eukaryotic cells. A growing body of evidenceimplicates PI3K and a lipid product of this enzyme, phosphatidylinositol(3,4,5)-triphosphate (hereinafter "PtdIns(3,4,5)P₃ "), as part of anovel and important second messenger system in cellular signaltransduction. The components of this novel PtdIns(3,4,5)P₃ -basedsignalling system appear to be independent of the previouslycharacterized signalling pathway based on inositol phospholipids, inwhich a phosphoinositidase C (PIC) hydrolyses PtdIns(4,5)P₂ to releasethe structurally distinct second messengers inositol(1,4,5)-triphosphate (Ins(1,4,5)P₃) and diacylglycerol.

Select extracellular agonists and growth factors will stimulateintracellular PI3K activity and cause the rapid and transientintracellular accumulation of PtdIns(3,4,5)P₃. Surprisingly, stimulationof a variety of different types of cell surface receptors, includingreceptor tyrosine kinases, receptors associated with src familynon-receptor tyrosine kinases, cytokine growth factors, and G proteincoupled receptors will all activate members of the PI3K family.(Reviewed in Stephens et al., 1993, Biochemica et Biophysica Acta,1179:27-75). For example, tyrosine kinase receptors which, whenactivated, result in increased accumulation of PtdIns(3,4,5)P₃ are thePDGF receptor, the EGF receptor, members of the FGF receptor family, theCSF-1 receptor, the insulin receptor, the IGF-1 receptor, and the NGFreceptor. Receptors associated with src family non-receptor tyrosinekinases which stimulate PtdIns(3,4,5)P₃ accumulation are the Il-2receptor, Il-3 receptor, mIgM receptor, the CD4 receptor, the CD2receptor, and the CD3/T cell receptor. Additionally, the cytokine Il-4receptor and the G protein linked thrombin receptor, ATP receptor, andthe fMLP receptor all stimulate the activity of a PI3K, resulting insubsequent PtdIns (3,4,5)P₃ accumulation. Thus, PtdIns(3,4,5)P₃ appearsto be a second messenger in extremely diverse signalling pathways.

Support for the proposition that PI3K activity and production ofPtdIns(3,4,5)P₃ is a physiological relevant pathway of signaltransduction for these diverse receptors is derived, inter alia, fromtwo different lines of experimental evidence: inhibition of PI3Kactivity by fungal metabolites and observations of direct proteinassociations. Wortmannin, a fungal metabolite, irreversibly inhibitsPI3K activity by binding covalently to the catalytic domain of thisenzyme. Inhibition of PI3K activity by wortmannin eliminates thesubsequent cellular response to the extracellular factor. For example,neutrophils respond to the chemokine fMet-Leu-Phe (fMLP) by stimulatingPI3K and synthesizing PtdIns(3,4,5)P₃. The synthesis correlates withactivation of the respiratory burst involved in neutrophil destructionof invading microorganisms. Treatment of neutrophils with wortmanninprevents the fMLP-induced respiratory burst response. Thelen et al.,1994, PNAS, U.S. Pat. No. 91:4960-4964. Indeed, these experiments withwortmannin, as well as other experimental evidence, shows that PI3Kactivity in cells of hematopoietic lineage, particularly neutrophils,monocytes, and other types of leukocytes, is involved in many of thenon-memory immune responses associated with acute and chronicinflammation.

PI3K enzymes interact directly with, and may be co-purified with,activated forms of several receptor tyrosine kinases. When purified,receptor tyrosine kinase associated PI3K was found to consist of 170-200kD heterodimers (Otsu et al., 1991, Cell 65:91-104, Pons et al., 1995,Mol. Cell. Biol. 15:4453-4465, Inukai et al., 1996, J. Biol. Chem.271:5317-5320) comprising a catalytic subunit and an adaptor (orregulatory) subunit.

Two different homologs of the catalytic subunit, p110α and p110β, havebeen described and cloned. The catalytic subunit, which irreversiblybinds wortmannin, tightly associates with one or other members of asmall family of highly related regulatory subunits, p55α, p55p1K, p85αand p85β, to form the 170-200 kD heterodimers. The known regulatorysubunits contain a large collection of protein:protein interactiondomains, including two SH2 domains (Cantley et al., 1991, Cell64:281-302).

The presence of the SH2 domains are thought to be responsible for thebinding and stimulation of PI3K heterodimers to activated receptortyrosine kinases. Activated receptors are phosphorylated at key tyrosineresidues within local consensus sequences preferred by the SH2 domainsfound in the 55-87 kD PI3K adaptors (Songyang et al., 1993, Cell72:767-778). Once the PI3K heterodimer binds, it directly activates thePI3K catalytic subunit (although this effect is relatively small invitro, Carpenter et al., 1993, J. Biol. Chem. 268:9478-9483, Backer etal., 1992, EMBO J. 11:3469-3479) and translocates the cytosolic PI3K toa source of its phospholipid substrate. The combination of these factorsleads to a surge in PtdIns(3,4,5)P₃ production. Clearly, these isoformsof PI3Ks (p100α/p110β/p55α, p55PIK) seem structurally adapted tofunction as dedicated signal transducers downstream ofreceptor-regulated tyrosine kinases, very like the way the τ-family ofPI-PLCs are regulated by receptor-sensitive tyrosine kinases (Lee andRhee, 1995, Current Biol. 7:193-189).

However, the p110/p85 sub-family of PI3Ks do not seem to be involved inthe production of PtdIns(3,4,5)P₃ that can occur as a result ofactivation of cell surface receptors which utilize heterotrimericGTPases to transduce their signals (e.g., fMLP, PAF, ATP, and thrombin).These types of cell surface receptors have been primarily described incells of hematopoietic origin whose activation is involved inflammatoryresponses of the immune system. Recent evidence has suggested that achromatographically distinct form of wortmannin-sensitive PI3K ispresent in U937 cells and neutrophils that possesses a native, relativemolecular mass of about 220 kD (Stephens et al., 1994, Cell 77:83-93).This PI3K activity can be specifically stimulated by Gβγ subunits, butnot Gα-GTP subunits. A similar PI3K activity has also been described inan Osteosarcoma cell line (Morris et al., 1995, Mol. Pharm. 48:532-539).Platelets also contain a Gβγ-sensitive PI3K, although it is unclearwhether this is a p85/p110 PI3K family member (Thomason et al., 1994, J.Biol. Chem. 269:16525-16528). It seemed likely that this poorlycharacterized, Gβγ-sensitive PI3K might be responsible for production ofPtdIns(3,4,5)P₃ in response to agonists like ATP, fMLP etc.

Stoyanov et al., (1995) have recently published the cloning andexpression of a wortmannin-sensitive PtdIns(4,5)P₂ -selective PI3K,termed p110γ, from a human bone marrow cDNA library. p110γ was amplifiedby PCR using primers designed to target potential PI3K's as well asPtdIns4-kinase's catalytic centers. It is clearly distinct from p110αand p111β, as it lacks, for example, an amino-terminus binding domainfor a member of the p85 adaptor family. p110γ was speculated to be thePI3K activity downstream of heterotrimeric GTPase-linked receptors onthe basis of its sensitivity to both Gα-GTP and Gβγ-subunits in vitroand its expression in myeloid-derived cells. Nevertheless, thishypothesis left several unresolved questions regarding the earlierbiochemical evidence which indicated that the Gβγ responsive PI3K wasnot stimulated by Gα-GTP subunits, and that it possessed a much greatermolecular mass of about 220 kD.

The effects of Gβγ subunits on p110γ were suggested to be mediated via aputative NH₂ -terminus pleckstrin homology (PH) domain. However, withthe description of an increasing number of Gβγ regulated effectors,mounting evidence suggests that PH domains do not represent a widelyused Gβγ binding domain. Recent work, using a panel of relatively smallpeptides based on the sequence of domains only found in theGβγ-activated adenylate cyclases (ACs 2 and 4) which specifically blockGβγ activation or inhibition of several effectors, has suggested theremay be some grounds for believing Gβγ subunits contain a widely usedeffector activating domain. Further, regions in different effectors thatinteract with this effector activating domain show significant sequencesimilarities. Hence a motif (Gln-X-X-Glu-Arg) within the domain in AC2highlighted by these peptide studies also appears in regions ofpotassium channels and β-ARKs already implicated in regulation by Gβγsubunits (Chen et al., 1995, Science 268:1166-1169). However, this motifis not replicated in all proteins known to be regulated by Gβγ subunits,and consequently sequence analysis cannot currently predict whether aprotein will be regulated by Gβγ subunits.

Identification of the mechanism by which PI3K activity is activated bycellular agonists which transduce their signals through G protein linkedreceptors is lacking. It is important to note that the vast majority ofagonists which activate the neutrophil respiratory burst involved in theinflammatory response will bind to G-protein-coupled receptors ratherthan receptor tyrosine kinases. Thus, the mechanism by which PI3K isregulated in response to these types of chemokines is likely to be verydifferent from regulation by growth factors which signal throughtyrosine kinases. The present invention is directed towards resolvingthis issue by the identification, purification, and cloning of a noveland specific form of PI3K which is activated by βγ subunits of trimericG-proteins.

III. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification,purification, and cloning of nucleotides that encode the trimeric Gprotein regulated PI3K, a novel protein that produces accumulation ofthe second messenger PtdIns(3,4,5)P₃ in response to activation of Gprotein-linked receptors. This novel G-protein regulated PI3K iscomprised of a catalytic subunit, p120, and a regulatory subunit, p110.The p120 catalytic subunit shares partial amino acid sequence homologywith the PI3K catalytic subunits p110α, p110β, and p110γ. p110, on theother hand, is a completely new protein with no identifiable homology toany other sequence previously available. In the absence of p101regulatory subunit, activated G protein subunits induce a mildstimulation of catalytic activity by the p120 catalytic subunit.However, in the presence of p101 subunit, the PI3K activity of thecatalytic subunit is stimulated over 100 fold by activated G proteins.

cDNAs encoding both porcine p101 and p120 and human p101 and p120 havebeen cloned and sequenced, and are described herein. The porcine p101and p120 cDNAs encode proteins of about 877 amino acids and about 1102amino acids, respectively (FIG. 2 and FIG. 4), while the human p101 andp120 cDNAs encode proteins of about 881 amino acids and about 1103 aminoacids, respectively (FIG. 11 and FIG. 13). Although the amino acidsequences of the p120 proteins are homologous to those of other knownPI3K catalytic subunits, the p120 cDNAs described herein diverge fromthe cDNAs encoding the known PI3K catalytic subunits, particularly at,for example, the carboxyl terminus (amino acid residues 1075 to 1102).

The p101 transcript is found primarily in cells of hematopoieticlineage. p120, and other PI3K catalytic subunit proteins, appear to havea far broader tissue and cell type distribution. Notably, the presenceof a trimeric G protein sensitive PI3K activity has only been found in alimited number of cells of hematopoietic lineage (e.g., neutrophils,platelets, etc.). Thus, the ability to activate PI3K enzymes in responseto stimulation of trimeric G protein linked receptors appears largelydependent on the presence of the p101 subunit.

The invention encompasses the following nucleotides, host cellsexpressing such nucleotides, and the expression products of suchnucleotides: (a) nucleotides that encode mammalian p101 and p120proteins, including the porcine p101 and p120 and the human p101 andp120, and the p101 and p120 gene products, including the porcine andhuman gene products; (b) nucleotides that encode portions of p101 andp120 that correspond to its functional domains, and the polypeptideproducts specified by such nucleotide sequences, including but notlimited to the p101 nucleotides encoding, for example, the p101 Gβγinteraction domain, or the catalytic subunit associating domain, oramino acid residues from about 1 to 160, 80-120, 161 to 263, 264 to 414,415 to 565, 566 to 706, 707-832, and/or 833 to 877, and p120 nucleotidesthat encode the p120 membrane binding domain, or the regulatory subunitdomain, or amino acid residues from about 173 to 302 and 310 to 315; (c)nucleotides that encode mutants of p101 and p120 in which all or a partof one of the domains is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences, including but notlimited to mutants of p101 wherein the nucleotides encoding the Gprotein interaction domain, or the catalytic subunit associating domain,or amino acid residues from about 1 to 160, 80-120, 161 to 263, 264 to414, 415 to 565, 566 to 706, 707-832, and/or 833 to 877 are deleted, andto mutants of p120 wherein the nucleotides encoding the membrane bindingdomain, or the regulatory subunit domain, or amino acid residues fromabout 173 to 302 and 310 to 315 are deleted; (d) nucleotides that encodefusion proteins containing the p101 protein or one of its domains (e.g.,the Gβγ interaction domain, or the catalytic subunit associating domain,or the domains described by amino acid residues from about 1 to 160,80-120, 161 to 263, 264 to 414, 415 to 565, 566 to 706, 707-832, and/or833 to 877), or the p120 protein or one of its domains (e.g., themembrane binding domain, or the regulatory subunit domain, or amino acidresidues from about 173 to 302 and 310 to 315) fused to anotherpolypeptide.

The invention also encompasses agonists and antagonists of G proteinregulated PI3K, including small molecules, large molecules, mutant p101proteins that compete with native p101, and antibodies, as well asnucleotide sequences that can be used to inhibit p101 gene expression(e.g., antisense and ribozyme molecules, and gene or regulatory sequencereplacement constructs) or to enhance p101 gene expression (e.g.,expression constructs that place the p101 gene under the control of astrong promoter system), and transgenic cells and animals that express ap101 transgene or "knock-outs" that do not express p101.

In addition, the present invention encompasses methods and compositionsfor the diagnostic evaluation, typing and prognosis of immune systemdisorders, including inflammation, and for the identification ofsubjects having a predisposition to such conditions. For example, p101nucleic acid molecules of the invention can be used as diagnostichybridization probes or as primers for diagnostic PCR analysis for theidentification of p101 gene mutations, allelic variations and regulatorydefects in the p101 gene. The present invention further provides fordiagnostic kits for the practice of such methods.

Further, the present invention also relates to methods for the use ofthe p101 gene and/or p120 gene products for the identification ofcompounds which modulate, i.e., act as agonists or antagonists, of Gprotein-regulated PI3K gene expression and or p101 and/or p120 geneproduct activity. Such compounds can be used as agents to control immunesystem disorders and, in particular, as therapeutic agents for thetreatment of inflammation disorders such as arthritis, septic shock,adult respiratory distress syndrome (ARDS), pneumonia, asthma,allergies, reperfusion injury, atherosclerosis, Alzheimer's disease, andcancer.

Still further, the invention encompasses methods and compositions forthe treatment of inflammation disorders such as arthritis, septic shock,adult respiratory distress syndrome (ARDS), pneumonia, asthma,allergies, reperfusion injury, atherosclerosis, Alzheimer's disease, andcancer. Such methods and compositions are capable of modulating thelevel of p101 gene expression and/or the level of p101 or p120 geneproduct activity.

This invention is based, in part, on the surprising discovery of a Gprotein stimulated PI3K activity in isolated neutrophils; thepurification and characterization of this activity as a heterodimericPI3K protein comprised of a p101 subunit and a p120 subunit; theidentification and cloning of p101 cDNA from libraries prepared fromporcine neutrophil mRNA, human monocyte mRNA, and human leukocyte mRNA;the identification and cloning of p120 cDNA from libraries prepared fromporcine neutrophil mRNA and human Leukocyte mRNA; characterization ofthe novel sequences; and studies of isolated recombinantly expressedp101 and p120 protein in insect cells, U937 cells, and Cos-7 cells.

A. DEFINITIONS

As used herein, the following terms, whether used in the singular orplural, will have the meanings indicated:

G protein-regulated PI3K: refers to a PI3K enzyme whose activity isstimulated by activated trimeric G proteins such as Gβγ subunits and/orGα-GTP subunits.

p101: means the regulatory subunit of the G protein-regulated PI3K, alsoknown as the adaptor subunit. p101 includes molecules that arehomologous to p101 or which bind to p120 and stimulate PI3K catalyticactivity in response to activation of trimeric G proteins. p101 fusionproteins having an N-terminal Glu tag (specifically MEEEEFMPMPMEF) arereferred to herein as (EE)101 fusion proteins, while p101 fusionproteins having an N-terminal myc epitope tag are referred to herein asmyc101 fusion proteins.

p101 nucleotides or coding sequences: means nucleotide sequencesencoding the p101 regulatory subunit protein, polypeptide or peptidefragments of p101 protein, or p101 fusion proteins. p101 nucleotidesequences encompass DNA, including genomic DNA (e.g. the p101 gene) orcDNA, or RNA.

120: means the catalytic subunit of the G protein-regulated PI3K.Polypeptides or peptide fragments of p120 protein are referred to asp120 polypeptides or p120 peptides. Fusions of p120, or p120polypeptides or peptide fragments to an unrelated protein are referredto herein as p120 fusion proteins. (EE)120 fusion proteins have theN-terminal Glu tag MEEEEFMPMEFSS. Functional equivalents of p120 referto a PI3K catalytic subunit protein which binds to the p101 regulatorysubunit with high affinity in vivo or in vitro. Other functionalequivalents of p120 are homologous catalytic subunits of a Gprotein-regulated PI3K, such as p117.

p120 nucleotides or coding sequences: means nucleotide sequencesencoding the p120 catalytic subunit protein, polypeptide or peptidefragments of p120 protein, or p120 fusion proteins. p120 nucleotidesequences encompass DNA, including genomic DNA (e.g. the p120 gene) orcDNA, or RNA.

IV. DESCRIPTION OF THE FIGURES

FIG. 1A, 1B, 1C, and 1D. Porcine p101 adaptor subunit nucleotidesequence. (SEQ ID NO:1)

FIG. 2. Deduced Porcine p101 amino acid sequence. (SEQ ID NO:2) Trypticpeptides identified by protein sequencing are underlined with a solidline.

FIGS. 3A, 3B and 3C. Porcine p120 catalytic subunit nucleotide sequence.(SEQ ID NO:3)

FIG. 4. Deduced Porcine p120 catalytic subunit sequence. (SEQ ID NO:4)Tryptic peptides identified by protein sequencing are underlined with asolid line. The region of divergence from the published amino acidsequence of p110γ is underscored with a broken line.

FIG. 5. Gβγ-sensitive PI3K in neutrophil cytosol is distinct to p85/p110PI3Ks. Aliquots of each of the fractions derived from a Q-sepharosechromatographic profile were either Western blotted and probed withαp85α or αp85β monoclonal antibodies and visualized with an HRP-linkedsecond antibody (upper panel in FIG. 5), or incubated with 100 nM ³H!-17-hydroxy-wortmannin, resolved by SDS-PAGE (6% acrylamide andfluorographed (lower panel in FIG. 5). Gβγ-sensitive PI3K activityeluted in fractions 20-24.

FIG. 6. Peak B After Final Mini Q Column Purification Contains aGβγ-sensitive PI3K Activity. The final purification product of the PeakB activity was analyzed after isolation from a Mini Q column in thepresence and absence of activated Gβγ subunits (βγ), a tyrosinephosphorylated peptide (YP-peptide), wortmannin, and/or Gα_(i) -GDPsubunits (α_(i) -GDP).

FIG. 7. Pharmacological and regulatory properties of free p120 andp101/p120 PI3Ks recombinantly expressed in Sf9 cells. Assays containedeither 7 nM p101/p120 or 36 nM p120 alone (final concentrations) whichwere incubated with various reagents; 10 mM NaF and 30 μM AlCl₃ (A/F); 1μM Gβγ subunits (βγ); 2 μM Gα-GDP; 100 nM wortmannin (W) or 50 μMtyrosine phosphorylated peptide (PY) for a total of 15 minutes (at 0°C.) prior to starting the assays by adding γ³² P!-ATP; ³²P!-incorporated into ³² P!-PtdIns(3,4,5)P₃ was quantified. The datashown are means (n=2).

FIG. 8. p101 can associate with a Gβγ-stimulated PI3K activity in U937cells. U937 cells were transiently transfected with mammalian expressionvectors encoding either (EE)120 or (EE)101, co-transfected incombination with mammalian expression vectors encoding myc101, myc120,or an irrelevant control myc fusion protein, as indicated. A total of 40μg of vector DNA was used. After co-transfection, the cells lysed,precleaned and immunoprecipitated with protein G sepharose covalentlycross-linked to α-(EE) monoclonal antibody as described more fully inthe Examples herein. The resulting immunoprecipitates were washed andGβγ-activated (1 μM, final concentration) PI3K activity was assayed. ThePI3K activity detected in immunoprecipitates from cells transfected withirrelevant (EE)-tagged protein, either with or without Gβγ wassubtracted from the data shown (these were means of 1896 dpm and 2862dpm in the absence and presence of Gβγ, respectively). Paralleltransfections labelled with ³⁵ S!-methionine showed the amount of ³⁵S!-p101 and ³⁵ S!-p120 in the immunoprecipitates fell by 40% when theywere co-transfected (data not shown).

FIG. 9. Regulation of p101/p120 by Gβγ in vivo. Cos-7 cells weretransfected with mammalian expression vectors encoding Gβγ subunit ("β₁γ₂ "), p101 ("101"), and/or p120 ("120"), as indicated. After 48 hoursof transient expression, cells from each transfection were eitherWestern blotted to determine "Relative Expression", or assayed for theaccumulation of PtdIns(3,4,5)P₃ accumulation (shaded bars). For Westernblotting, samples were probed with an α-(myc) monoclonal antibody toquantitate the expression of the various (myc)-tagged proteins. Resultsare given relative to the expression obtained in the presence of all 4key vectors; the absolute levels of (myc)-p120 and (myc)-p101 were verysimilar and about 10× greater than that of (myc)-γ₂. Parallel batches ofcells were labelled with ³² P!-Pi; after 90 minutes lipids wereextracted, deacylated and water-soluble head groups resolved byanion-exchange HPLC and quantitated by liquid scintillation counting.Data shown are means (n=2) +/- ranges. Data for ³² P!-PtdIns(3,4,5)P₃are above the irrelevant DNA control (972±41 dpm).

FIGS. 10A and 10B. Human p101 adaptor subunit nucleotide sequence. (SEQID NO:11)

FIG. 11. Deduced Human p101 amino acid sequence. (SEQ ID NO:12)

FIGS. 12A and 12B. Human p120 catalytic subunit nucleotide sequence.(SEQ ID NO:13)

FIG. 13. Deduced Human p120 catalytic subunit sequence. (SEQ ID NO:14)

V. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification, purification, andcloning of a specific form of PI3K which is activated by βγ subunits oftrimeric G-proteins. p101, described for the first time herein, is anovel subunit of the G-protein regulated PI3K. Also described herein isthe identification, cloning, and correct sequence of p120, the catalyticsubunit of the trimeric G-protein regulated PI3K.

PI3K are enzymes which phosphorylate phosphatidyl inositols at the 3dposition to generate the intracellular signaling moleculePtdIns(3,4,5)P₃. Although it has been shown that PI3K's are induced in avariety of cell types upon stimulation of tyrosine kinase receptors,PI3K's which are activated by trimeric G-protein linked receptors haveonly been detected in a limited number of hematopoietic lineage derivedcells such as platelets, monocytes, and leukocytes. Interestingly,accumulation of PtdIns(3,4,5)P₃ in these cells is associated with manyof the immune responses involved in acute and chronic inflammation. Forexample, neutrophils are activated by, inter alia, the chemokine fMLPwhich is released in response to infection by microorganisms. Thisagonist binds to a pertussis toxin sensitive G protein coupled receptorand activates the neutrophil respiratory burst, resulting in superoxideproduction and cytotoxicity. The respiratory burst response correlateswith a rapid and transient intracellular increase in PI3K activity.

Studies in neutrophils demonstrates that wortmannin, which selectivelyinhibits the catalytic component of PI3K's, causes a shutdown ofsuperoxide production. Since the vast majority of agonists whichactivate this respiratory burst bind to G protein-linked receptors, theG protein regulated PI3K activity represents a candidate for a dominanteffector pathway whose inhibition will reduce the destructive cellulareffects of inflammation. However, wortmannin is not a clinicallyappropriate inhibitor of this pathway since wortmannin inhibits all PI3Kcatalytic activities, including those involved in other cellularpathways such as growth factors.

Reported herein is the discovery that the G-protein regulated PI3K iscomposed of two subunits: the catalytic subunit (p110γ, p117 or 120);and the p101 regulatory subunit. The ability of PI3K to respond to cellsurface receptors which stimulate the release of Gβγ subunits fromactivated trimeric G-proteins is largely dependent on the presence ofthe p101 regulatory subunit. Although catalytic subunits exhibit a smallstimulation (approximately 1.7 fold) of PI3K activity in vitro in thepresence of Gβγ subunits, addition of p101 proteins will increase Gβγstimulation of PI3K activity 100 fold. Neutralization of p110, removalof p110, or interference of p101 binding to its binding partners, willrender the cells incapable of generating greatly increased levels of theintracellular signal PtdIns(3,4,5)P₃ in response to activated Gβγsubunits. Thus, the limited distribution of the p101 subunit in bonemarrow derived cells appears to be the critical factor in the cell typespecificity of this response. Furthermore, p101 contains no significanthomologies with any other identified sequence. This discovery makesp101, and the p101/p120 complex, the target of choice to inhibit,activate, or modulate G protein-activated PI3K with minimal non-specificeffects.

The invention also encompasses the use of p101 and p120 nucleotides,p101 and p120 proteins and peptides, as well as antibodies to p101 andp120 (which can, for example, act as p101 or p120 agonists orantagonists), antagonists that inhibit G protein-activated PI3K activityor expression, or agonists that activate G protein-activated PI3Kactivity or increase its expression in the treatment of hematopoieticlineage cell activation disorders, including, but not limited to immuneresponses associated with acute and chronic inflammation, in animalssuch as humans. For example, antagonists of Gβγ-activated PI3K will beuseful in the treatment of arthritis, including rheumatoid arthritis,septic shock, adult respiratory distress syndrome (ARDS), pneumonia,asthma and other lung conditions, allergies, reperfusion injury,atherosclerosis and other cardiovascular diseases, Alzheimer's disease,and cancer, to name just a few inflammatory disorders. In addition, p101nucleotides and p101 regulatory subunits, as well as p120 nucleotidesand p120 catalytic subunits, are useful for the identification ofcompounds effective in the treatment of hematopoietic lineage cellactivation disorders involving G protein-activated PI3Ks.

Further, the invention encompasses the use of p101 and p120 nucleotides,p101 and p120 proteins and peptides, as well as antibodies to p101 andp120 in the diagnosis of hematopoietic lineage cell activationdisorders. The diagnosis of a p101 regulatory subunit or p120 catalyticsubunit abnormality in a patient, or an abnormality in the G proteinactivated PI3K signal transduction pathway, will also assist in devisinga proper treatment or therapeutic regimen.

In particular, the invention described in the subsections belowencompasses p101 regulatory subunit, polypeptides or peptidescorresponding to functional domains of the p101 regulatory subunit(e.g., the catalytic subunit association domain, or the domain whichinteracts with activated G proteins), mutated, truncated or deleted p101regulatory subunits (e.g. a p101 regulatory subunit with one or morefunctional domains or portions thereof deleted), p101 regulatory subunitfusion proteins (e.g. a p101 regulatory subunit or a functional domainof p101 regulatory subunit, fused to an unrelated protein or peptidesuch as an epitope tag, i.e., the myc epitope), nucleotide sequencesencoding such products, and host cell expression systems that canproduce such p101 regulatory subunit products.

Additionally, the invention encompasses p120 catalytic subunit proteins,polypeptides, functional domains of the p120 subunit (e.g., thecatalytic domain), mutated, truncated or deleted p120 subunit proteins,p120 fusion proteins, nucleotide sequences encoding such products, andhost cell expression systems that can produce such p120 catalyticsubunit products.

The invention also encompasses antibodies and anti-idiotypic antibodies(including Fab fragments), antagonists and agonists of the Gprotein-activated PI3K, as well as compounds or nucleotide constructsthat inhibit expression of the p101 or p120 gene (transcription factorinhibitors, antisense and ribozyme molecules, or gene or regulatorysequence replacement constructs), or promote expression of p101regulatory subunit (e.g., expression constructs in which p101 codingsequences are operatively associated with expression control elementssuch as promoters, promoter/enhancers, etc.). The invention also relatesto host cells and animals genetically engineered to express the humanp101 regulatory subunit (or mutants thereof) or to inhibit or"knock-out" expression of the animal's endogenous p101 regulatorysubunit.

Further, the invention particularly encompasses antagonists whichprevent the association of p101 regulatory subunits with their bindingpartners, including p120 and other PI3K catalytic subunit proteins suchas p117 and p110γ, as well as activated trimeric G protein proteins,including Gβγ subunits.

The p101 regulatory subunit proteins or peptides, p101 regulatorysubunit fusion proteins, p101 nucleotide sequences, antibodies,antagonists and agonists can be useful for the detection of mutant p101regulatory subunits or inappropriately expressed p101 regulatorysubunits for the diagnosis of immune disorders. The p101 and p120subunit proteins or peptides, p101 and p120 subunit fusion proteins,p101 and l120 nucleotide sequences, host cell expression systems,antibodies, antagonists, agonists and genetically engineered cells andanimals can be used for screening for drugs effective in the treatmentof such immune disorders. The use of engineered host cells and/oranimals may offer an advantage in that such systems allow not only forthe identification of compounds that bind to the p101 regulatorysubunit, but can also identify compounds that affect the signaltransduced by the activated p101 regulatory subunit, specifically,production of the intracellular signaling molecule PtdIns(3,4,5)P₃.

Finally, the p101 regulatory subunit protein products and fusion proteinproducts, antibodies and anti-idiotypic antibodies (including Fabfragments), antagonists or agonists (including compounds that modulatesignal transduction which may act on downstream targets in the p101regulatory subunit signal transduction pathway) can be used for therapyof such diseases. For example, nucleotide constructs encoding functionalp101 regulatory subunits, mutant p101 regulatory subunits, as well asantisense and ribozyme molecules can be used in "gene therapy"approaches for the modulation of p101 regulatory subunit expressionand/or activity in the treatment of hematopoietic lineage cellactivation disorders. Thus, the invention also encompassespharmaceutical formulations and methods for treating hematopoieticlineage cell activation disorders.

The invention is based, in part, on the surprising discovery of novelPI3K enzymes in porcine neutrophils. Like other PI3K proteins, theseenzymes 3-phosphorylate PtdIns, PtdIns4P and PtdIns(4,5)P₂ substrates,and were completely inhibited by 100 nM wortmannin. However, unlike thepreviously described PI3K p85/p101 protein complexes, the PI3K activitywas stimulated over 100 fold by incubation with Gβγ subunits. Additionof Gα-GDP subunits could inhibit this Gβγ activation. Furthermore, thePI3K activity was not stimulated by phosphorylated tyrosine peptides.When purified, two distinct heterodimeric protein complex wereidentified: a p117/p101 complex and a 120/p101 complex. Peptidesequencing revealed that the p101 protein was identical in each complex.The p120 and p117 proteins were homologous with the exception of theamino terminus (see Examples below). Porcine p101 and p120 cDNAs werethen cloned using degenerate probes based upon the peptide sequence toscreen an expression library of cDNAs synthesized from porcineneutrophil mRNA. Human p101 and p120 were cloned using probes derivedfrom the porcine cDNA sequences to screen human monocyte and leukocytecDNA libraries. While sequence analysis of the porcine and humansubunits revealed that the p120 proteins are homologous to previouslycloned PI3K catalytic subunits (although they diverge significantly atthe extreme carboxyl terminus), the p101 proteins are unrelated to anysequence in the databases.

Comparison of the amino acid sequences of the porcine and human homologsof p101 and p120 reveals a high degree of conservation between the twospecies. This high degree of conservation between two mammalian speciessuggests that a similar degree of conservation probably exists amongmammalian species in general. In the same way that the porcine sequenceswere used to clone their human homologs, one skilled in the art canclone p101 and p120 homologs from other mammalian species using thedisclosed human and/or porcine sequences and the methods described inthis specification.

Experiments described herein expressing p101 and/or p120 fusion proteinsin insect cells demonstrated that p101 binds tightly to p120 in a 1:1molar stoichiometry. Free purified p120 exhibited PI3K activity whichwas insensitive to the presence of Gα subunits and tyrosinephosphorylated peptides, and only mildly stimulated by Gβγ subunits.However, when bound to p110, the PI3K activity of p120 was stimulated100 fold by the presence of Gβγ subunits. When p101 was expressed alonein insect cells, p101 did not exhibit PI3K activity.

An interesting result occurred when p101 was expressed as a taggedfusion protein in human U937 cells. When this recombinantly expressedporcine p101 was immunoprecipitated via the fusion protein tag, theseimmunoprecipitants did contain G protein regulated PI3K activity.Coexpression of p120 with p101 slightly decreased the amount of PI3Kactivity that could be immunoprecipitated. These results indicated thehuman U937 cells contained a PI3K catalytic subunit which could bind toand be activated by the porcine p101 regulatory subunit. These resultsdemonstrate that along with their highly conserved amino acid sequences,homologs from different mammalian species resemble one anotherfunctionally and structurally.

Further, transient transfections were performed in Cos-7 cells, which donot normally stimulate PI3K activity in response to activatedG-proteins, with constructs encoding p101, p120 and/or Gβγ subunits.Transfection of a construct which expressed p120 only producedsignificant increases in cellular PtdIns(3,4,5)P₃ levels in a Gβγdependent fashion when co-expressed in the presence of p101.

Various aspects of the invention are described in greater detail in thesubsections below.

A. THE p101 AND p120 GENES

The cDNA sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ IDNO:2) of porcine p101 regulatory subunit are shown in FIGS. 1 and 2,respectively. A relatively common allelic variation occurs at amino acidresidue 483 in the open reading frame; a serine may be replaced by aglycine at this position.

The cDNA sequence (SEQ ID NO:11) and deduced amino acid sequence (SEQ IDNO:12) of human p101 regulatory subunit are shown in FIGS. 10 and 11,respectively.

The nucleotide sequence encoding the first 733 amino acids of theporcine p101 regulatory subunit, corresponding to the first 736 aminoacids of the human subunit, is believed to be sufficient to bind thecatalytic subunit. However, without the 145 carboxyl terminal aminoacids, this truncated p101 bound to p120 will not stimulate PI3Kactivity in response to Gβγ subunits. Therefore, the catalytic subunitassociating domain is believed to be contained within the first 732amino acids (735 for human), and the carboxyl terminal amino acids 733to 877 (736 to 880) could be involved in the response to Gβγ subunits.Other domains of porcine p101 are described by amino acid residues fromabout 1 to 160, 80-120, 161 to 263, 264 to 414, 415 to 565, 566 to 706,707-832, and/or 833 to 877. The corresponding domains of human p101 aredescribed by amino acid residues from about 1 to 160, 80-120, 161 to263, 264 to 416, 417 to 567, 568 to 709, 710-835, and/or 836 to 880. Thenucleotide sequences which encode amino acid residues from about 161 to263 define a pleckstrin homology ("PH") domain (PROSITE:PS50003). PHdomains may be involved in binding of proteins to Gβγ subunits (Touharaet al., 1994, J. Biol. Chem. 269:10217) and to membrane phospholipids(see Shaw, 1996, Bioessays 18:35-46). Thus, the PH domain of p101 may beinvolved in both of these events.

The nucleotide sequences encoding amino acids 1 to 160 of p101 may beresponsible for binding to the p120 catalytic subunit. When this regionof the p101 protein is analyzed for secondary structure, it is predictedto have a self-contained alternating α-helix/β-sheet structure. Withinthis structure is found homology to a "WW domain" (Staub et al., 1996,Structure 4:495-499 and TIBS 21:161-163). This WW domain may bind to aproline-rich domain found within the N-terminus of p120 protein(residues 310 to 315). Thus, the WW domain of p101 may be involved inmediating the interaction between the regulatory subunit and thecatalytic subunit.

The cDNA sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ IDNO:4) of porcine p120 are shown in FIGS. 3 and 4, respectively. The cDNAsequence (SEQ ID NO:13) and deduced amino acid sequence (SEQ ID NO:14)of human p120 are shown in FIGS. 12 and 13, respectively. A crypticthrombin cleavage site is present after the first approximately 40 aminoacid residues. The truncated p120 protein lacking these approximately 40amino terminal residues is still able to bind to p101, but PI3K activityis reduced approximately 20 to 30%. In other words, a protein having theamino acid sequence of the section of SEQ ID NO:4 (porcine p120 deducedamino acid sequence) extending from about amino acid residue 41 toresidue 1102 retains the functionality of full-length p120. Based on thehigh degree of sequence homology between porcine and human p120, aprotein having the amino acid sequence of the section of SEQ ID NO:14(human p120 deduced amino acid sequence) extending from about amino acidresidue 41 to residue 1102 would also retain the functionality offull-length p120. Although the p120 protein was highly homologous to thepreviously cloned p110γ protein reported by Stoyanov et al. (1995), theextreme C-terminus of p120 diverges from the reported p110γ protein atamino acid residue 1075; thus, the last 28 amino acid residues of p120have not been published in reports of any homologous protein. As notedabove, the nucleotides encoding a proline-rich region including p120residues 310 to 315 may be involved in the interaction between p120 andp110. Additionally, the nucleotides encoding p120 amino acid residuesfrom about 173 to 302 define a weak PH domain which may be a candidatefor membrane binding and/or Gβγ subunit interaction of the p101/p120complex.

Data presented in the working examples, infra, demonstrate that the p120cDNA encodes the catalytic subunit of the Gβγ-activated PI3K. The p101cDNA encodes a novel regulatory subunit protein which binds to the p120subunit. This heterodimer 3-phosphorylates PtdIns, PtdIns4P andPtdIns(4,5)P₂ in response to activation of trimeric G-protein linkedreceptors.

The p101 nucleotide sequences of the invention include: (a) the DNAsequence shown in FIG. 1 or FIG. 10 or contained in the cDNA clonepCMV3mycp101 as deposited with the American Type Culture Collection(ATCC) under accession number 97636; (b) nucleotide sequence thatencodes the amino acid sequence shown in FIG. 2 or FIG. 11, or the p101regulatory subunit amino acid sequence encoded by the cDNA clonepCMV3mycp101 as deposited with the ATCC; (c) any nucleotide sequencethat hybridizes to the complement of the DNA sequence shown in FIG. 1 orFIG. 10 or contained in the cDNA clone pCMV3mycp101, as deposited withthe ATCC under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc., and John Wiley & sons, Inc., NewYork, at p. 2.10.3) and encodes a functionally equivalent gene product;and (d) any nucleotide sequence that hybridizes to the complement of theDNA sequences that encode the amino acid sequence shown in FIG. 1 orFIG. 10 or contained in the cDNA clone pCMV3mycp101, as deposited withthe ATCC, under less stringent conditions, such as moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet which still encodes a functionally equivalent p101gene product. Functional equivalents of the p101 regulatory subunitinclude naturally occurring p101 regulatory subunit present in otherspecies, and mutant p101 regulatory subunits whether naturally occurringor engineered which retain at least some of the functional activities ofp101 (i.e., binding to the p120 or p117 catalytic subunit, stimulationof catalytic activity in response to Gβγ subunits, and/or interactionwith Gβγ subunits). The invention also includes degenerate variants ofsequences (a) through (d).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thenucleotide sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides ("oligos"), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may encode or act as p101 antisensemolecules, useful, for example, in p101 gene regulation (for and/or asantisense primers in amplification reactions of p101 gene nucleic acidsequences). With respect to p101 gene regulation, such techniques can beused to regulate, for example, inflammatory immune responses. Further,such sequences may be used as part of ribozyme and/or triple helixsequences, also useful for p101 gene regulation. Still further, suchmolecules may be used as components of diagnostic methods whereby, forexample, the presence of a particular p101 allele responsible forcausing an inflammatory response, such as arthritis, may be detected.

In addition to the p101 nucleotide sequences described above, fulllength p101 cDNA or gene sequences present in the same species and/orhomologs of the p101 gene present in other species can be identified andreadily isolated, without undue experimentation, by molecular biologicaltechniques well known in the art. Experimental evidence described hereinindicates that the p101 proteins are conserved in different mammalianspecies. For example, human p101 was cloned from monocyte and leukocytelibraries using probes derived from the porcine p101 cDNA sequence, andthe deduced amino acid sequences of the human and porcine were found tobe highly conserved. The structural and functional similarity of the twohomologs is evidenced by experiments showing that when porcine p101 wasexpressed in U937 cells, the porcine p101 bound to a human homolog ofthe catalytic subunit. Additionally, members of the tyrosine kinaseregulated PI3K family of proteins, for example the p110/p85 PI3K, arealso conserved among different mammalian species. Therefore, homologs ofp101 and p120 can be isolated from a variety of mammalian cells known orsuspected to contain a trimeric G-protein regulated PI3K, particularlycells of hematopoietic origin, and more particularly platelets,monocytes, leukocytes, osteoclasts, and neutrophils.

The identification of homologs of p101 in related species can be usefulfor developing animal model systems more closely related to humans forpurposes of drug discovery. For example, expression libraries of cDNAssynthesized from neutrophil mRNA derived from the organism of interestcan be screened using labeled catalytic subunit derived from thatspecies, e.g., a p120, p117, or p110γ catalytic subunit fusion protein.Alternatively, such cDNA libraries, or genomic DNA libraries derivedfrom the organism of interest can be screened by hybridization using thenucleotides described herein as hybridization or amplification probes.Furthermore, genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of thep101 gene product can also be identified via similar techniques. In thecase of cDNA libraries, such screening techniques can identify clonesderived from alternatively spliced transcripts in the same or differentspecies.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of the human orporcine p101 nucleotide sequence, as shown in FIGS. 1 and 10,respectively. The hybridization washing conditions used should be of alower stringency when the cDNA library is derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. With respect to the cloning of a mammalian p101 homolog, usingporcine and/or human p101 probes, for example, hybridization can, forexample, be performed at 65° C. overnight in Church's buffer (7% SDS,250 mM NaHPO₄, 2 μM EDTA, 1% BSA). Washes can be done with 2×SSC, 0.1%SDS at 65° C. and then at 0.1×SSC, 0.1% SDS at 65° C.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

Alternatively, the labeled p101 nucleotide probe may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. The identification andcharacterization of human genomic clones is helpful for designingdiagnostic tests and clinical protocols for treating hematopoieticlineage cell activation disorders in human patients. For example,sequences derived from regions adjacent to the intron/exon boundaries ofthe human gene can be used to design primers for use in amplificationassays to detect mutations within the exons, introns, splice sites (e.g.splice acceptor and/or donor sites), etc., that can be used indiagnostics.

Further, a p101 gene homolog may be isolated from nucleic acid of theorganism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the p101 gene product disclosed herein. The templatefor the reaction may be cDNA obtained by reverse transcription of mRNAprepared from, for example, human or non-human cell lines or cell types,such as neutrophils, known or suspected to express a p101 gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a p101 gene. The PCRfragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular source (i.e., one known, orsuspected, to express the p101 gene, such as, for example, neutrophilsor other types of leukocytes). A reverse transcription reaction may beperformed on the RNA using an oligonucleotide primer specific for themost 5' end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be "tailed" withguanines using a standard terminal transferase reaction, the hybrid maybe digested with RNAase H, and second strand synthesis may then beprimed with a poly-C primer. Thus, cDNA sequences upstream of theamplified fragment may easily be isolated. For a review of cloningstrategies which may be used, see e.g., Sambrook et al., 1989, supra.

The p101 gene sequences may additionally be used to isolate mutant p101gene alleles. Such mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes to thesymptoms of hematopoietic lineage cell activation disorders such asinflammation. Mutant alleles and mutant allele products may then beutilized in the therapeutic and diagnostic systems described below.Additionally, such p101 gene sequences can be used to detect p101 generegulatory (e.g., promoter or promotor/enhancer) defects which canaffect hematopoietic lineage cell activation.

A cDNA of a mutant p101 gene may be isolated, for example, by using PCR,a technique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from cells known or suspectedto be expressed in an individual putatively carrying the mutant p101allele, and by extending the new strand with reverse transcriptase. Thesecond strand of the cDNA is then synthesized using an oligonucleotidethat hybridizes specifically to the 5' end of the normal gene. Usingthese two primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant p101 allele to that of the normal p101 allele, themutation(s) responsible for the loss or alteration of function of themutant p101 gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry the mutant p101allele, or a cDNA library can be constructed using RNA from a cell typeknown, or suspected, to express the mutant p101 allele. The normal p101gene or any suitable fragment thereof may then be labeled and used as aprobe to identify the corresponding mutant p101 allele in suchlibraries. Clones containing the mutant p101 gene sequences may then bepurified and subjected to sequence analysis according to methods wellknown to those of skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a cell type known, orsuspected, to express a mutant p101 allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant cell type may be expressed and screenedusing standard antibody screening techniques in conjunction withantibodies raised against the normal p101 gene product, as described,below, in Section 5.3. (For screening techniques, see, for example,Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", ColdSpring Harbor Press, Cold Spring Harbor.) Additionally, screening can beaccomplished by screening with labeled fusion proteins, such as, forexample, the (EE)120 or the myc120 fusion proteins. In cases where ap101 mutation results in an expressed gene product with altered function(e.g., as a result of a missense or a frameshift mutation), a polyclonalset of antibodies to p101 regulatory subunit are likely to cross-reactwith the mutant p101 regulatory subunit gene product. Library clonesdetected via their reaction with such labeled antibodies can be purifiedand subjected to sequence analysis according to methods well known tothose of skill in the art.

The invention also encompasses nucleotide sequences that encode mutantp101 regulatory subunits, peptide fragments of the p101 regulatorysubunit, truncated p101 regulatory subunits, and p101 regulatory subunitfusion proteins. These include, but are not limited to nucleotidesequences encoding mutant p101 regulatory subunits described in section5.2 infra; polypeptides or peptides corresponding to the catalyticbinding, or Gβγ subunit binding domains of the p101 regulatory subunitor portions of these domains; truncated p101 regulatory subunits inwhich one or two of the domains is deleted, or a truncated,nonfunctional p101 regulatory subunit. Nucleotides encoding fusionproteins may include but are not limited to full length p101 regulatorysubunit, truncated p101 regulatory subunit or peptide fragments of p101regulatory subunit fused to an unrelated protein or peptide, such as forexample, an epitope tag which aids in purification or detection of theresulting fusion protein; or an enzyme, fluorescent protein, luminescentprotein which can be used as a marker.

The invention also encompasses (a) DNA vectors that contain any of theforegoing p101 regulatory subunit coding sequences and/or theircomplements (i.e., antisense); (b) DNA expression vectors that containany of the foregoing p101 regulatory subunit coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences; and (c) genetically engineered hostcells that contain any of the foregoing p101 regulatory subunit codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the baculoviruspromoter, cytomegalovirus hCMV immediate early gene promoter, the earlyor late promoters of SV40 adenovirus, the lac system, the trp system,the TAC system, the TRC system, the major operator and promoter regionsof phage A, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters of the yeast α-mating factors.

Finally, the invention also encompasses nucleotides encoding p120subunit proteins including the newly described carboxyl terminus of thiscatalytic subunit, deletion variants of p120 subunit proteins,nucleotides which hybridize to these nucleotides under highly stringentconditions and which encode functionally equivalent products, includingthe cDNA clone pCMV3mycp120 deposited with the ATCC under accessionnumber 97637, allelic variants of p120 (e.g., mutant alleles or thenaturally occurring alleles such as the allelic variation at amino acidresidue 483), and equivalent p120 nucleotides from different organismsisolated as described above for the p101 nucleotides of the invention.Additionally, the invention encompasses expression vectors and hostcells for the recombinant production of p120.

B. p101 AND p120 PROTEINS AND POLYPEPTIDES

p101 regulatory subunit and p120 catalytic subunit, polypeptides andpeptide fragments, mutated, truncated or deleted forms of the p101regulatory subunit and/or p101 regulatory subunit fusion proteins andthe p120 catalytic subunit and/or p120 catalytic subunit fusion proteinscan be prepared for a variety of uses, including but not limited to thegeneration of antibodies, as reagents in diagnostic assays, theidentification of other cellular gene products involved in theregulation of hematopoietic lineage cell activation, as reagents inassays for screening for compounds that can be used in the treatment ofhematopoietic lineage cell activation disorders, and as pharmaceuticalreagents useful in the treatment of hematopoietic lineage cellactivation disorders related to Gβγ-activated PI3K.

FIGS. 2 and 11 show the amino acid sequences of porcine and human p101regulatory subunit protein, respectively. FIGS. 4 and 13 show the aminoacid sequence of porcine and human p120 catalytic subunit protein,respectively. The broken line on FIG. 4 underscores the region of p120which diverges from the published sequences of the PI3K catalyticsubunits p110α, p110β, and p110γ.

The p101 regulatory subunit sequence begins with a methionine in a DNAsequence context consistent with a translation initiation site. Thepredicted molecular mass of both porcine and human p101 regulatorysubunits is 97 kD.

The p101 regulatory subunit amino acid sequences of the inventioninclude the amino acid sequences shown in FIG. 2 (SEQ ID NO:2) or FIG. 4(SEQ ID NO:12), or the amino acid sequence encoded by the cDNA clonepCMV3mycp101, as deposited with the ATCC. Further, p101 regulatorysubunits of other species are encompassed by the invention. In fact, anyp101 regulatory subunit protein encoded by the p101 nucleotide sequencesdescribed above, are within the scope of the invention.

The p120 catalytic subunit amino acid sequences of the invention includethe cDNA clone pCMV3mycp120, as deposited with the ATCC. The inventionalso encompasses p120 catalytic subunits of other species, and the p120proteins encoded by the p120 nucleotide sequences described above in theprevious section.

The invention also encompasses proteins that are functionally equivalentto the p101 regulatory subunit encoded by the nucleotide sequencesdescribed above, as judged by any of a number of criteria, including butnot limited to the ability to bind catalytic subunit, the bindingaffinity for catalytic subunit, the ability to stimulate PI3K activityof the catalytic subunit in response to activated trimeric G proteins,the resulting biological effect of catalytic subunit binding andresponse to activation of trimeric G proteins, e.g., signaltransduction, a change in cellular metabolism (e.g., generation ofPtdIns(3,4,5)P₃) or change in phenotype when the p101 regulatory subunitequivalent is present in an appropriate cell type (such as thesuperoxide burst in neutrophils). Such functionally equivalent p101regulatory subunit proteins include but are not limited to additions orsubstitutions of amino acid residues within the amino acid sequenceencoded by the p101 nucleotide sequences described, above, but whichresult in a silent change, thus producing a functionally equivalent geneproduct. Amino acid substitutions may be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Similarly, theinvention also encompasses functional equivalents of p120 protein, asdescribed above.

While random mutations can be made to p101 DNA (using random mutagenesistechniques well known to those skilled in the art) and the resultingmutant p101 regulatory subunits tested for activity, site-directedmutations of the p101 coding sequence can be engineered (usingsite-directed mutagenesis techniques well known to those skilled in theart) to generate mutant p101 regulatory subunits with increasedfunction, e.g., higher binding affinity for catalytic subunit, and/orgreater signalling capacity; or decreased function, e.g., lower bindingaffinity for catalytic subunit, and/or decreased signal transductioncapacity. For example, porcine p101 amino acid sequence may be alignedwith that of human p101 regulatory subunit. Mutant p101 regulatorysubunits can be engineered so that regions of interspecies identity aremaintained, whereas the variable residues are altered, e.g., by deletionor insertion of an amino acid residue(s) or by substitution of one ormore different amino acid residues. Conservative alterations at thevariable positions can be engineered in order to produce a mutant p101regulatory subunit that retains function; e.g., catalytic subunitbinding affinity or activated G protein transduction capability or both.Non-conservative changes can be engineered at these variable positionsto alter function, e.g., catalytic subunit binding affinity or signaltransduction capability, or both. Alternatively, where alteration offunction is desired, deletion or non-conservative alterations of theconserved regions can be engineered. One of skill in the art may easilytest such mutant or deleted p101 regulatory subunits for thesealterations in function using the teachings presented herein.

Other mutations to the p101 coding sequence can be made to generate p101regulatory subunits that are better suited for expression, scale up,etc. in the host cells chosen. For example, the triplet code for eachamino acid can be modified to conform more closely to the preferentialcodon usage of the host cell's translational machinery.

Peptides corresponding to one or more domains (or a portion of a domain)of the p101 regulatory subunit (e.g., the p120 binding domain, the Gprotein interacting domain, or the domains defined by amino acidresidues from about 1 to 150, 151 to 300, 301 to 450, 451 to 600,601-732 (porcine) or 601-735 (human), and 733-877 (porcine) or 736-881(human)), truncated or deleted p101 regulatory subunits (e.g., p101regulatory subunit in which portions of one or more of the above domainsare deleted) as well as fusion proteins in which the full length p101regulatory subunit, a p101 regulatory subunit peptide or truncated p101regulatory subunit is fused to an unrelated protein are also within thescope of the invention and can be designed on the basis of the p101nucleotide and p101 regulatory subunit amino acid sequences disclosed inthis Section and above. Such fusion proteins include but are not limitedto fusions to an epitope tag (such as is exemplified in the Examplesbelow); or fusions to an enzyme, fluorescent protein, or luminescentprotein which provide a marker function.

While the p101 regulatory subunit polypeptides and peptides can bechemically synthesized (e.g., see Creighton, 1983, Proteins: Structuresand Molecular Principles, W.H. Freeman & Co., N.Y.), large polypeptidesderived from the p101 regulatory subunit and the full length p101regulatory subunit itself may advantageously be produced by recombinantDNA technology using techniques well known in the art for expressingnucleic acid containing p101 gene sequences and/or coding sequences.Such methods can be used to construct expression vectors containing thep101 nucleotide sequences described above and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel etal., 1989, supra. Alternatively, RNA capable of encoding p101 nucleotidesequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in"Oligonucleotide Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to expressthe p101 nucleotide sequences of the invention. Where the p101regulatory subunit peptide or polypeptide is a soluble derivative thepeptide or polypeptide can be recovered from the culture, i.e., from thehost cell in cases where the p101 regulatory subunit peptide orpolypeptide is not secreted, and from the culture media in cases wherethe p101 regulatory subunit peptide or polypeptide is secreted by thecells. However, such engineered host cells themselves may be used insituations where it is important not only to retain the structural andfunctional characteristics of the p101 regulatory subunit, but to assessbiological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing p101 nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the p101 nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the p101 sequences;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing p101 nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3, U937) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the p101gene product being expressed. For example, when a large quantity of sucha protein is to be produced, for the generation of pharmaceuticalcompositions of p101 regulatory subunit protein or for raisingantibodies to the p101 regulatory subunit protein, for example, vectorswhich direct the expression of high levels of fusion protein productsthat are readily purified may be desirable. Such vectors include, butare not limited, to the E. coli expression vector pUR278 (Ruther et al.,1983, EMBO J. 2:1791), in which the p101 coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 264:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). If the inserted sequence encodes a relatively smallpolypeptide (less than 25 kD), such fusion proteins are generallysoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety. Alternatively, if the resulting fusionprotein is insoluble and forms inclusion bodies in the host cell, theinclusion bodies may be purified and the recombinant protein solubilizedusing techniques well known to one of skill in the art.

In an insect system, Autoqrapha californica nuclear polyhidrosis virus(AcNPV) may be used as a vector to express foreign genes. (E.g., seeSmith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).In a specific embodiment described below, Sf9 insect cells are infectedwith a baculovirus vectors expressing either a 6× HIS-tagged p120construct, or an (EE)-tagged p101 construct.

In mammalian host cells, a number of viral-based expression systems maybe utilized. Specific embodiments described more fully below expresstagged p101 or p120 cDNA sequences using a CMV promoter to transientlyexpress recombinant protein in U937 cells or in Cos-7 cells.Alternatively, retroviral vector systems well known in the art may beused to insert the recombinant expression construct into host cells. Forexample, retroviral vector systems for transducing hematopoietic cellsare described in published PCT applications WO 96/09400 and WO 94/29438.

In cases where an adenovirus is used as an expression vector, the p101nucleotide sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe p101 gene product in infected hosts. (E.g., See Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signalsmay also be required for efficient translation of inserted p101nucleotide sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire p101 gene or cDNA,including its own initiation codon and adjacent sequences, is insertedinto the appropriate expression vector, no additional translationalcontrol signals may be needed. However, in cases where only a portion ofthe p101 coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript may be used. Such mammalianhost cells include but are not limited to CHO, VERO, BHK, HeLa, COS,MDCK, 293, 3T3, WI38, and U937 cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe p101 sequences described above may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the p101gene product. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the p101 gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

The p101 gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees may be used to generate p101transgenic animals.

Any technique known in the art may be used to introduce the p101transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry thep101 transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the p101 gene transgene beintegrated into the chromosomal site of the endogenous p101 gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous p101 gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous p101 gene. Inthis way, the expression of the endogenous p101 gene may also beeliminated by inserting non-functional sequences into the endogenousgene. The transgene may also be selectively introduced into a particularcell type, thus inactivating the endogenous p101 gene in only that celltype, by following, for example, the teaching of Gu et al. (Gu et al.,1994, Science 265: 103-106). The regulatory sequences required for sucha cell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant p101 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof cell type samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of p101 gene-expressing tissue, may alsobe evaluated immunocytochemically using antibodies specific for the p101transgene product, as described below.

C. ANTIBODIES TO p101 AND p120 PROTEINS

Antibodies that specifically recognize one or more epitopes of p101regulatory subunit, or epitopes of conserved variants of p101 regulatorysubunit, or peptide fragments of the p101 regulatory subunit are alsoencompassed by the invention. Also encompassed by the invention areantibodies which recognize one or more epitopes of the p120 protein,particularly, the novel carboxyl terminus. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab')₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of the p101 regulatory subunit or p120 in a biological sampleand may, therefore, be utilized as part of a diagnostic or prognostictechnique whereby patients may be tested for abnormal amounts of theseproteins. Such antibodies may also be utilized in conjunction with, forexample, compound screening schemes, as described, below, in Section5.5, for the evaluation of the effect of test compounds on expressionand/or activity of the p101 or p120 gene products. Additionally, suchantibodies can be used in conjunction with the gene therapy techniquesdescribed, below, in Section 5.6, to, for example, evaluate the normaland/or engineered p101 regulatory subunit-expressing cells prior totheir introduction into the patient. Such antibodies may additionally beused as a method for the inhibition of abnormal p101 regulatory subunitor p120 activity. Thus, such antibodies may, therefore, be utilized aspart of inflammatory disorder treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the p101 regulatory subunit, a p101 regulatory subunitpeptide, truncated p101 regulatory subunit polypeptides, functionalequivalents of the p101 regulatory subunit or mutants of the p101regulatory subunit. Additionally, host animals may be immunized byinjection with p120 catalytic subunit or peptides of the p120 subunit.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjutants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjutants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of "chimericantibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a porcine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against p101 gene products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab')₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to the p101 regulatory subunit or the p120 catalytic subunitcan, in turn, be utilized to generate anti-idiotype antibodies that"mimic" the p101 regulatory subunit or p120 subunit, respectively, usingtechniques well known to those skilled in the art. (See, e.g., Greenspan& Bona, 1993, FASEB J 7(5) :437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to the p101regulatory subunit and competitively inhibit the binding of catalyticsubunit to the p101 regulatory subunit can be used to generateanti-idiotypes that "mimic" the p101 regulatory subunit and, therefore,bind and neutralize catalytic subunit. Such neutralizing anti-idiotypesor Fab fragments of such anti-idiotypes can be used in therapeuticregimens to neutralize catalytic subunit and reduce inflammation.

D. DIAGNOSIS OF HEMATOPOIETIC CELL ACTIVATION DISORDERS

A variety of methods can be employed for the diagnostic and prognosticevaluation of hematopoietic lineage cell activation disorders, includinginflammatory disorders, and for the identification of subjects having apredisposition to such disorders.

Such methods may, for example, utilize reagents such as the p101 andp120 nucleotide sequences described above, and p101 regulatory subunitand p120 antibodies, as described, in Section 5.3. Specifically, suchreagents may be used, for example, for: (1) the detection of thepresence of p101 or p120 gene mutations, or the detection of eitherover- or under-expression of p101 or p120 mRNA relative to thenon-hematopoietic lineage cell activation disorder state; (2) thedetection of either an over- or an under-abundance of p101 or p120 geneproduct relative to the non-hematopoietic lineage cell activationdisorder state; and (3) the detection of perturbations or abnormalitiesin the signal transduction pathway mediated by p101 regulatory subunitand p120 catalytic subunit.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific p101 orp120 nucleotide sequence or p101 or p120 regulatory subunit antibodyreagent described herein, which may be conveniently used, e.g., inclinical settings, to diagnose patients exhibiting hematopoietic lineagecell activation disorder abnormalities.

For the detection of p101 or p120 mutations, any nucleated cell can beused as a starting source for genomic nucleic acid. For the detection ofp101 or p120 gene expression or p101 or p120 gene products, any celltype or tissue in which the p101 or p120 gene is expressed, such as, forexample, neutrophil cells, may be utilized.

Nucleic acid-based detection techniques are described, below, in Section5.4.1. Peptide detection techniques are described, below, in Section5.4.2.

1. DETECTION OF THE p101 GENE AND TRANSCRIPTS

Mutations within the p101 or p120 genes can be detected by utilizing anumber of techniques. Nucleic acid from any nucleated cell can be usedas the starting point for such assay techniques, and may be isolatedaccording to standard nucleic acid preparation procedures which are wellknown to those of skill in the art.

DNA may be used in hybridization or amplification assays of biologicalsamples to detect abnormalities involving gene structure, includingpoint mutations, insertions, deletions and chromosomal rearrangements.Such assays may include, but are not limited to, Southern analyses,single stranded conformational polymorphism analyses (SSCP), and PCRanalyses.

Such diagnostic methods for the detection of p101 or p120 gene-specificmutations can involve for example, contacting and incubating nucleicacids including recombinant DNA molecules, cloned genes or degeneratevariants thereof, obtained from a sample, e.g., derived from a patientsample or other appropriate cellular source, with one or more labelednucleic acid reagents including recombinant DNA molecules, cloned genesor degenerate variants thereof, as described above, under conditionsfavorable for the specific annealing of these reagents to theircomplementary sequences within the p101 or p120 gene. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid molecule hybrid. The presence of nucleicacids which have hybridized, if any such molecules exist, is thendetected. Using such a detection scheme, the nucleic acid from the celltype or tissue of interest can be immobilized, for example, to a solidsupport such as a membrane, or a plastic surface such as that on amicrotiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described aboveare easily removed. Detection of the remaining, annealed, labeled p101or p120 nucleic acid reagents is accomplished using standard techniqueswell-known to those in the art. The p101 or p120 gene sequences to whichthe nucleic acid reagents have annealed can be compared to the annealingpattern expected from a normal gene sequence in order to determinewhether a gene mutation is present.

Alternative diagnostic methods for the detection of p101 or p120 genespecific nucleic acid molecules, in patient samples or other appropriatecell sources, may involve their amplification, e.g., by PCR (theexperimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No.4,683,202), followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. The resultingamplified sequences can be compared to those which would be expected ifthe nucleic acid being amplified contained only normal copies of thep101 or p120 gene in order to determine whether a gene mutation exists.

Additionally, well-known genotyping techniques can be performed toidentify individuals carrying p101 or p120 gene mutations. Suchtechniques include, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

The level of p101 or p120 gene expression can also be assayed bydetecting and measuring p101 or p120 transcription. For example, RNAfrom a cell type or tissue known, or suspected to express the p101 orp120 gene, such as hematopoietic lineage cells, especially myeloid cellsand platelets, may be isolated and tested utilizing hybridization or PCRtechniques such as are described, above. The isolated cells can bederived from cell culture or from a patient. The analysis of cells takenfrom culture may be a necessary step in the assessment of cells to beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the p101 or p120gene. Such analyses may reveal both quantitative and qualitative aspectsof the expression pattern of the p101 or p120 gene, including activationor inactivation of p101 or p120 gene expression.

2. DETECTION OF THE p101 GENE PRODUCTS

Antibodies directed against wild type or mutant p101 or p120 geneproducts or conserved variants or peptide fragments thereof, which arediscussed, above, in Section 5.3, may also be used as hematopoieticlineage cell activation disorder diagnostics and prognostics, asdescribed herein. Such diagnostic methods, may be used to detectabnormalities in the level of p101 or p120 gene expression, orabnormalities in the structure and/or temporal, tissue, cellular, orsubcellular location of the p101 regulatory subunit, and may beperformed in vivo or in vitro, such as, for example, on biopsy tissue.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to contain cells express the p101 or p120gene, such as, for example, neutrophil cells which have infiltrated aninflamed tissue. The protein isolation methods employed herein may, forexample, be such as those described in Harlow and Lane (Harlow, E. andLane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety. The isolated cells can be derivedfrom cell culture or from a patient. The analysis of cells taken fromculture may be a necessary step in the assessment of cells that could beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the p101 or p120gene.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.3, useful in the present invention may beused to quantitatively or qualitatively detect the presence of p101 orp120 gene products or conserved variants or peptide fragments thereof.This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below, this Section)coupled with light microscopic, flow cytometric, or fluorimetricdetection.

The antibodies (or fragments thereof) or fusion or conjugated proteinsuseful in the present invention may, additionally, be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immuno assays, for in situ detection of p101 or p120 gene productsor conserved variants or peptide fragments thereof, or for catalyticsubunit binding (in the case of labeled catalytic subunit fusionprotein).

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody orfusion protein of the present invention. The antibody (or fragment) orfusion protein is preferably applied by overlaying the labeled antibody(or fragment) onto a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of the p101or p120 gene product, or conserved variants or peptide fragments, butalso its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for p101 or p120 gene products orconserved variants or peptide fragments thereof will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cells which have been incubatedin cell culture, in the presence of a detectably labeled antibodycapable of identifying p101 or p120 gene products or conserved variantsor peptide fragments thereof, and detecting the bound antibody by any ofa number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled p101 regulatorysubunit or p120 subunit antibody or fusion protein. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody or fusion protein. The amount of bound label on solidsupport may then be detected by conventional means.

"Solid phase support or carrier" is intended to encompass any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of p101 regulatory subunit or p120subunit antibody or fusion protein may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

With respect to antibodies, one of the ways in which the antibody can bedetectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay(ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological AssociatesQuarterly Publication, Walkersville, Md.); Voller et al., 1978, J. Clin.Pathol. 31:507-520; Butler, 1981, Meth. Enzymol. 73:482-523; Maggio(ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawaet al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). Theenzyme which is bound to the antibody will react with an appropriatesubstrate, preferably a chromogenic substrate, in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect p101 regulatory subunitthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

E. SCREENING ASSAYS FOR COMPOUNDS THAT MODULATE G-PROTEIN ACTIVATED PI3KEXPRESSION OR ACTIVITY

The following assays are designed to identify compounds that interactwith (e.g., bind to) p101 regulatory subunit or the p120 catalyticsubunit, compounds that interact with (e.g., bind to) intracellularproteins that interact with p101 regulatory subunit and/or the p120catalytic subunit, compounds that interfere with the interaction of p101regulatory subunit with the p120 catalytic subunit or with otherintracellular proteins involved in G protein stimulated PI3K mediatedsignal transduction, and to compounds which modulate the activity ofp101 or p120 gene (i.e., modulate the level of p101 or p120 geneexpression) or modulate the level of p101 or p120. Assays mayadditionally be utilized which identify compounds which bind to p101 orp120 gene regulatory sequences (e.g., promoter sequences) and which maymodulate p101 or p120 gene expression. See e.g., Platt, K. A., 1994, J.Biol. Chem. 269:28558-28562, which is incorporated herein by referencein its entirety.

The compounds which may be screened in accordance with the inventioninclude but are not limited to peptides, antibodies and fragmentsthereof, prostaglandins, lipids and other organic compounds (e.g.,terpines, peptidomimetics) that bind to the p101 regulatory subunit orp120 catalytic subunit and either mimic the activity triggered by thenatural ligand (i.e., agonists) or inhibit the activity triggered by thenatural ligand (i.e., antagonists); as well as peptides, antibodies orfragments thereof, and other organic compounds that mimic the p101regulatory subunit or the p120 catalytic subunit (or a portion thereof)and bind to and "neutralize" natural ligand.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), andcombinatorial chemistry-derived molecular library peptides made of D-and/or L-configuration amino acids, phosphopeptides (including, but notlimited to members of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell72:767-778); antibodies (including, but not limited to, polyclonal,monoclonal, humanized, anti-idiotypic, chimeric or single chainantibodies, and FAb, F(ab')₂ and FAb expression library fragments, andepitopebinding fragments thereof); and small organic or inorganicmolecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able togain entry into an appropriate cell (e.g., in the neutrophil) and affectthe expression of the p101 or p120 gene or some other gene involved inthe p101 regulatory subunit signal transduction pathway (e.g., byinteracting with the regulatory region or transcription factors involvedin gene expression); or such compounds that affect the activity of thep101 regulatory subunit, e.g., by inhibiting or enhancing the binding ofp101 to the catalytic subunit of the PI3K or the binding of p101 to someother intracellular factor involved in the p101 regulatory subunitsignal transduction pathway, such as, for example, Gβγ.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate p101 regulatory subunit or p120 catalytic subunit expression oractivity. Having identified such a compound or composition, the activesites or regions are identified. Such active sites might typically bethe binding partner sites, such as, for example, the interaction domainsof the p120 catalytic subunit with p101 regulatory subunit itself. Theactive site can be identified using methods known in the art including,for example, from the amino acid sequences of peptides, from thenucleotide sequences of nucleic acids, or from study of complexes of therelevant compound or composition with its natural ligand. In the lattercase, chemical or X-ray crystallographic methods can be used to find theactive site by finding where on the factor the complexed ligand isfound.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intramolecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modelling can be used to completethe structure or improve its accuracy. Any recognized modelling methodmay be used, including parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential G protein activated PI3Kmodulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites ofp120 catalytic subunit, p101 regulatory subunit, and relatedtransduction and transcription factors will be apparent to those ofskill in the art.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modelling of drugs interactive withspecific proteins, such as Rotivinen et al., 1988, Acta PharmaceuticalFennica 97:159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinalyand Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perryand Davies, OSAR: Quantitative Structure-Activity Relationships in DrugDesign pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989,Proc. R. Soc. Lond. 236:125-140 and 141-162; and, with respect to amodel receptor for nucleic acid components, Askew et al., 1989, J. Am.Chem. Soc. 111:1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these areprimarily designed for application to drugs specific to particularproteins, they can be adapted to design of drugs specific to regions ofDNA or RNA, once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of the p101or p120 gene product, and for ameliorating hematopoietic lineage cellactivation disorders. Assays for testing the effectiveness of compounds,identified by, for example, techniques such as those described inSection 5.5.1 through 5.5.3, are discussed, below, in Section 5 5.5.4.

1. In vitro SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO p101 REGULATORYSUBUNIT

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) p101 regulatory subunit or p120catalytic subunit. Compounds identified may be useful, for example, inmodulating the activity of wild type and/or mutant p101 or p120 geneproducts; may be utilized in screens for identifying compounds thatdisrupt normal p101 regulatory subunit/catalytic subunit interactions;or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to thep101 regulatory subunit involves preparing a reaction mixture of thep101 regulatory subunit and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The p101 regulatory subunit species used can vary dependingupon the goal of the screening assay. For example, where agonists of thenatural ligand are sought, the full length p101 regulatory subunit, or afusion protein containing the p101 regulatory subunit fused to a proteinor polypeptide that affords advantages in the assay system (e.g.,labeling, isolation of the resulting complex, etc.) can be utilized.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the p101regulatory subunit protein, polypeptide, peptide or fusion protein orthe test substance onto a solid phase and detecting p101 regulatorysubunit/test compound complexes anchored on the solid phase at the endof the reaction. In one embodiment of such a method, the p101 regulatorysubunit reactant may be anchored onto a solid surface, and the testcompound, which is not anchored, may be labeled, either directly orindirectly. In another embodiment of the method, a p101 regulatorysubunit protein anchored on the solid phase is complexed with labeledcatalytic subunit such as p120. Then, a test compound could be assayedfor its ability to disrupt the association of the p101/p120 complex.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for p101regulatory subunit protein, polypeptide, peptide or fusion protein, orthe catalytic subunit protein or fusion protein, or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

2. ASSAYS FOR INTRACELLULAR PROTEINS THAT INTERACT WITH THE p101 OR p120PROTEINS

Any method suitable for detecting protein-protein interactions may beemployed for identifying intracellular proteins that interact with p101regulatory subunit and/or the catalytic subunit p120. Among thetraditional methods which may be employed are co-immunoprecipitation,crosslinking and co-purification through gradients or chromatographiccolumns of cell lysates or proteins obtained from cell lysates and thep101 regulatory subunit to identify proteins in the lysate that interactwith the p101 regulatory subunit. For these assays, the p101 regulatorysubunit component used can be a full length p101 regulatory subunit, ora truncated peptide. Similarly, the component may be a p120 catalyticsubunit, or a complex of the p101 regulatory subunit with the p120catalytic subunit. Once isolated, such an intracellular protein can beidentified and can, in turn, be used, in conjunction with standardtechniques, to identify proteins with which it interacts. For example,at least a portion of the amino acid sequence of an intracellularprotein which interacts with the p101 regulatory subunit, PI3K(p101/p120 complex), or p120 catalytic subunit, can be ascertained usingtechniques well known to those of skill in the art, such as via theEdman degradation technique. (See, e.g., Creighton, 1983, "Proteins:Structures and Molecular Principles", W.H. Freeman & Co., N.Y.,pp.34-49). The amino acid sequence obtained may be used as a guide forthe generation of oligonucleotide mixtures that can be used to screenfor gene sequences encoding such intracellular proteins. Screening maybe accomplished, for example, by standard hybridization or PCRtechniques. Techniques for the generation of oligonucleotide mixturesand the screening are well-known. (See, e.g., Ausubel, supra., and PCRProtocols: A Guide to Methods and Applications, 1990, Innis, M. et al.,eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result in the simultaneousidentification of genes which encode the intracellular proteinsinteracting with p101 regulatory subunit and/or the p120 catalyticsubunit and/or the PI3K. These methods include, for example, probingexpression, libraries, in a manner similar to the well known techniqueof antibody probing of λgt11 libraries, using labeled p101 regulatorysubunit protein, or a p101 regulatory subunit polypeptide, peptide orfusion protein, e.g., a p101 regulatory subunit polypeptide or p101regulatory subunit domain fused to a marker (e.g., an enzyme, fluor,luminescent protein, or dye), or an Ig-Fc domain.

One method which detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to a p101nucleotide sequence encoding p101 regulatory subunit, a p101 regulatorysubunit polypeptide, peptide or fusion protein, and the other plasmidconsists of nucleotides encoding the transcription activator protein'sactivation domain fused to a cDNA encoding an unknown protein which hasbeen recombined into this plasmid as part of a cDNA library. TheDNA-binding domain fusion plasmid and the cDNA library are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., HBS or lacZ) whose regulatory region contains thetranscription activator's binding site. Either hybrid protein alonecannot activate transcription of the reporter gene; the DNA-bindingdomain hybrid cannot because it does not provide activation function,and the activation domain hybrid cannot because it cannot localize tothe activator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the "bait"gene product. By way of example, and not by way of limitation, p101regulatory subunit may be used as the bait gene product. Total genomicor cDNA sequences are fused to the DNA encoding an activation domain.This library and a plasmid encoding a hybrid of a bait p101 gene productfused to the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait p101 gene sequence, such as the open reading frame of p101 (or adomain of p101), as depicted in FIG. 1 can be cloned into a vector suchthat it is translationally fused to the DNA encoding the DNA-bindingdomain of the GAL4 protein. These colonies are purified and the libraryplasmids responsible for reporter gene expression are isolated. DNAsequencing is then used to identify the proteins encoded by the libraryplasmids.

A cDNA library of the cell line from which proteins that interact withbait p101 gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transfected along withthe bait p101 gene-GAL4 fusion plasmid into a yeast strain whichcontains a lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait p101 gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies which express HIS3 can be detected by their growthon petri dishes containing semi-solid agar based media lackinghistidine. The cDNA can then be purified from these strains, and used toproduce and isolate the bait p101 gene-interacting protein usingtechniques routinely practiced in the art.

3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH p101 REGULATORYSUBUNIT/INTRACELLULAR MACROMOLECULE INTERACTION

The macromolecules that interact with the p101 regulatory subunit arereferred to, for purposes of this discussion, as "binding partners".These binding partners are likely to be involved in the p101 regulatorysubunit signal transduction pathway, and therefore, in the role of p101regulatory subunit in hematopoietic lineage cell activation regulation.Known binding partners are catalytic subunits of the PI3K kinase such asp120, p117, and perhaps certain p110 proteins. Other binding partnersare likely to be activated trimeric G proteins such as Gβγ subunits, andor lipids. Therefore, it is desirable to identify compounds thatinterfere with or disrupt the interaction of such binding partners withp101 which may be useful in regulating the activity of the p101regulatory subunit and thus control hematopoietic lineage cellactivation disorders associated with p101 regulatory subunit activity.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the p101 regulatory subunit andits binding partner or partners involves preparing a reaction mixturecontaining p101 regulatory subunit protein, polypeptide, peptide orfusion protein as described in Sections 5.5.1 and 5.5.2 above, and thebinding partner under conditions and for a time sufficient to allow thetwo to interact and bind, thus forming a complex. In order to test acompound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of the p101 regulatory subunit moiety and itsbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe p101 regulatory subunit moiety and the binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the p101 regulatory subunitand the interactive binding partner. Additionally, complex formationwithin reaction mixtures containing the test compound and normal p101regulatory subunit protein may also be compared to complex formationwithin reaction mixtures containing the test compound and a mutant p101regulatory subunit. This comparison may be important in those caseswherein it is desirable to identify compounds that disrupt interactionsof mutant but not normal p101 regulatory subunits.

The assay for compounds that interfere with the interaction of the p101regulatory subunit and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the p101 regulatory subunit moiety product or thebinding partner onto a solid phase and detecting complexes anchored onthe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction by competition can beidentified by conducting the reaction in the presence of the testsubstance; i.e., by adding the test substance to the reaction mixtureprior to or simultaneously with the p101 regulatory subunit moiety andinteractive binding partner. Alternatively, test compounds that disruptpreformed complexes, e.g. compounds with higher binding constants thatdisplace one of the components from the complex, can be tested by addingthe test compound to the reaction mixture after complexes have beenformed. The various formats are described briefly below.

In a heterogeneous assay system, either the p101 regulatory subunitmoiety or the interactive binding partner, is anchored onto a solidsurface, while the non-anchored species is labeled, either directly orindirectly. In practice, microtiter plates are conveniently utilized.The anchored species may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the p101 or p120 geneproduct or binding partner and drying. Alternatively, an immobilizedantibody specific for the species to be anchored may be used to anchorthe species to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notprelabeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the p101 regulatorysubunit moiety and the interactive binding partner is prepared in whicheither the p101 regulatory subunit or its binding partners is labeled,but the signal generated by the label is quenched due to formation ofthe complex (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein whichutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances which disrupt p101 regulatorysubunit/intracellular binding partner interaction can be identified.

In a particular embodiment, a p101 regulatory subunit fusion can beprepared for immobilization. For example, the p101 regulatory subunit ora peptide fragment, e.g., corresponding to the CD, can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive binding partner can bepurified and used to raise a monoclonal antibody, using methodsroutinely practiced in the art and described above, in Section 5.3. Thisantibody can be labeled with the radioactive isotope ¹²⁵ I, for example,by methods routinely practiced in the art. In a heterogeneous assay,e.g., the GST-p101 regulatory subunit fusion protein can be anchored toglutathione-agarose beads. The interactive binding partner can then beadded in the presence or absence of the test compound in a manner thatallows interaction and binding to occur. At the end of the reactionperiod, unbound material can be washed away, and the labeled monoclonalantibody can be added to the system and allowed to bind to the complexedcomponents. The interaction between the p101 or p120 gene product andthe interactive binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-p101 regulatory subunit fusion protein and theinteractive binding partner can be mixed together in liquid in theabsence of the solid glutathione-agarose beads. The test compound can beadded either during or after the species are allowed to interact. Thismixture can then be added to the glutathione-agarose beads and unboundmaterial is washed away. Again the extent of inhibition of the p101regulatory subunit/binding partner interaction can be detected by addingthe labeled antibody and measuring the radioactivity associated with thebeads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the p101 regulatory subunit and/or the interactive or binding partner(in cases where the binding partner is a protein), in place of one orboth of the full length proteins. Any number of methods routinelypracticed in the art can be used to identify and isolate the bindingsites. These methods include, but are not limited to, mutagenesis of thegene encoding one of the proteins and screening for disruption ofbinding in a coimmunoprecipitation assay. Compensating mutations in thegene encoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a p101 gene product can beanchored to a solid material as described, above, by making a GST-p101regulatory subunit fusion protein and allowing it to bind to glutathioneagarose beads. The interactive binding partner can be labeled with aradioactive isotope, such as ³⁵ S, and cleaved with a proteolytic enzymesuch as trypsin. Cleavage products can then be added to the anchoredGST-p101 fusion protein and allowed to bind. After washing away unboundpeptides, labeled bound material, representing the intracellular bindingpartner binding domain, can be eluted, purified, and analyzed for aminoacid sequence by well-known methods Peptides so identified can beproduced synthetically or fused to appropriate facilitative proteinsusing recombinant DNA technology.

4. ASSAYS FOR IDENTIFICATION OF COMPOUNDS THAT AMELIORATE INFLAMMATORYDISORDERS

Compounds, including but not limited to binding compounds identified viaassay techniques such as those described, above, in Sections 5.5.1through 5.5.3, can be tested for the ability to ameliorate immune systemdisorder symptoms, including inflammation. The assays described abovecan identify compounds which affect p101 regulatory subunit activity(e.g., compounds that bind to the p101 regulatory subunit, inhibitbinding of the natural ligands, and either activate signal transduction(agonists) or block activation (antagonists), and compounds that bind toa natural ligand of the p101 regulatory subunit and neutralize theligand activity); or compounds that affect p101 or p120 gene activity(by affecting p101 or p120 gene expression, including molecules, e.g.,proteins or small organic molecules, that affect or interfere withsplicing events so that expression of the full length or the truncatedform of the p101 regulatory subunit can be modulated). However, itshould be noted that the assays described herein can also identifycompounds that modulate p101 regulatory subunit signal transduction(e.g., compounds which affect downstream signaling events, such asinhibitors or enhancers of activities which participate in transducingthe PtdIns(4,5)P₃ signal which is generated by catalytic subunit bindingto the p101 regulatory subunit). The identification and use of suchcompounds which affect another step in the p101 regulatory subunitsignal transduction pathway in which the p101 or p120 gene and/or p101or p120 gene product is involved and, by affecting this same pathway maymodulate the effect of p101 regulatory subunit on the development ofhematopoietic lineage cell activation disorders are within the scope ofthe invention. Such compounds can be used as part of a therapeuticmethod for the treatment of hematopoietic lineage cell activationdisorders.

The invention encompasses cell-based and animal model-based assays forthe identification of compounds exhibiting such an ability to amelioratehematopoietic lineage cell activation disorder symptoms. Such cell-basedassay systems can also be used as the standard to assay for purity andpotency of the natural ligand, catalytic subunit, includingrecombinantly or synthetically produced catalytic subunit and catalyticsubunit mutants.

Cell-based systems can be used to identify compounds which may act toameliorate hematopoietic lineage cell activation disorder symptoms. Suchcell systems can include, for example, recombinant or non-recombinantcells, such as cell lines, which express the p101 and/or p120 gene. Forexample leukocyte cells, or cell lines derived from leukocyte cells canbe used. In addition, expression host cells (e.g., COS cells, CHO cells,fibroblasts, Sf9 cells) genetically engineered to express a functionalp101/p120 PI3K and to respond to activation by the natural ligand Gβγsubunits, e.g., as measured by a chemical or phenotypic change,induction of another host cell gene, change in intracellular messengerlevels (e.g., PtdIns(3,4,5)P₃, etc.), can be used as an end point in theassay.

In utilizing such cell systems, cells may be exposed to a compoundsuspected of exhibiting an ability to ameliorate hematopoietic lineagecell activation disorder symptoms, at a sufficient concentration and fora time sufficient to elicit such an amelioration of hematopoieticlineage cell activation disorder symptoms in the exposed cells. Afterexposure, the cells can be assayed to measure alterations in theexpression of the p101 or p120 gene, e.g., by assaying cell lysates forp101 or p120 mRNA transcripts (e.g., by Northern analysis) or for p101or p120 protein expressed in the cell; compounds which regulate ormodulate expression of the p101 or p120 gene are valuable candidates astherapeutics. Alternatively, the cells are examined to determine whetherone or more hematopoietic lineage cell activation disorder-like cellularphenotypes has been altered to resemble a more normal or more wild typephenotype, or a phenotype more likely to produce a lower incidence orseverity of disorder symptoms. Still further, the expression and/oractivity of components of the signal transduction pathway of which p101regulatory subunit is a part, or the activity of the p101 regulatorysubunit signal transduction pathway itself can be assayed.

For example, after exposure of the cells, cell lysates can be assayedfor the presence of increased levels of the second messengerPtdIns(3,4,5)P₃, compared to lysates derived from unexposed controlcells. The ability of a test compound to inhibit production of secondmessenger in these assay systems indicates that the test compoundinhibits signal transduction initiated by p101 regulatory subunitactivation. The cell lysates can be readily assayed using anion-exchangeHPLC. Alternatively, levels of superoxide production or O₂ may beassayed by monitoring chemiluminescence from horseradish-peroxidasecatalyzed luminol oxidation as described in Wymann et al., 1987, Anal.Biochem. 165:371-378, incorporated herein by reference in its entirety.Finally, a change in cellular adhesion of intact cells may be assayedusing techniques well known to those of skill in the art.

In addition, animal-based hematopoietic lineage cell activation disordersystems, which may include, for example, mice, may be used to identifycompounds capable of ameliorating hematopoietic lineage cell activationdisorder-like symptoms. Such animal models may be used as test systemsfor the identification of drugs, pharmaceuticals, therapies andinterventions which may be effective in treating such disorders. Forexample, animal models may be exposed to a compound, suspected ofexhibiting an ability to ameliorate hematopoietic lineage cellactivation disorder symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of hematopoietic lineagecell activation disorder symptoms in the exposed animals. The responseof the animals to the exposure may be monitored by assessing thereversal of disorders associated with hematopoietic lineage cellactivation disorders such as inflammation. With regard to intervention,any treatments which reverse any aspect of hematopoietic lineage cellactivation disorder-like symptoms should be considered as candidates forhuman hematopoietic lineage cell activation disorder therapeuticintervention. Dosages of test agents may be determined by derivingdose-response curves, as discussed below.

F. THE TREATMENT OF DISORDERS ASSOCIATED WITH STIMULATION OF G-PROTEINACTIVATED PI3K, INCLUDING INFLAMMATORY DISORDERS

The invention also encompasses methods and compositions for modifyinghematopoietic lineage cell activation and treating hematopoietic lineagecell activation disorders, including inflammatory disorders. Forexample, by decreasing the level of p101 gene expression, and/or p101regulatory subunit gene activity, and/or downregulating activity of thep101 regulatory subunit pathway (e.g., by interfering with theinteraction of p101 regulatory subunit with the p120 catalytic subunit,or by targeting downstream signalling events), the response of leukocytecells to factors which activate trimeric G protein associated receptors,such as cytokines, may be reduced and the symptoms of chronicinflammatory diseases ameliorated. Conversely, the response of leukocytecells to activation of G protein associated receptors may be augmentedby increasing p101 regulatory subunit activity. For example, suchaugmentation may serve to boost the response of the immune system toinfections. Different approaches are discussed below.

1. INHIBITION OF p101 ADAPTOR EXPRESSION OR p101 ADAPTOR ACTIVITY TOREDUCE G PROTEIN ACTIVATED PI3K ACTIVITY AND REDUCE INFLAMMATION

Any method which neutralizes catalytic subunit or inhibits expression ofthe p101 or p120 gene (either transcription or translation) can be usedto reduce the inflammatory response. Such approaches can be used totreat inflammatory response disorders such as arthritis, includingrheumatoid arthritis, septic shock, adult respiratory distress syndrome(ARDS), pneumonia, asthma and other lung conditions, allergies,reperfusion injury, atherosclerosis and other cardiovascular diseases,Alzheimer's disease, and cancer, to name just a few inflammatorydisorders.

In one embodiment, immuno therapy can be designed to reduce the level ofendogenous p101 or p120 gene expression, e.g., using antisense orribozyme approaches to inhibit or prevent translation of p101 or p120mRNA transcripts; triple helix approaches to inhibit transcription ofthe p101 or p120 gene; or targeted homologous recombination toinactivate or "knock out" the p101 or p120 gene or its endogenouspromoter.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to p101 or p120 regulatory subunit mRNA.The antisense oligonucleotides will bind to the complementary p101 orp120 mRNA transcripts and prevent translation. Absolute complementarity,although preferred, is not required. A sequence "complementary" to aportion of an RNA, as referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex. In the case of double-stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the message,e.g., the 5' untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3' untranslatedsequences of mRNAs have recently shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5'- or3'- non- translated, non-coding regions of the p101 or p120 shown inFIG. 1 and FIG. 3 could be used in an antisense approach to inhibittranslation of endogenous p101 or p120 mRNA. Oligonucleotidescomplementary to the 5' untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention. Whetherdesigned to hybridize to the 5'-, 3'- or coding region of p101regulatory subunit mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988), orhybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988,Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5'-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In another embodiment, the antisense oligonucleotide comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al., 1988, Nucl. Acids Res. 16:3209.Methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451).

While antisense nucleotides complementary to the p101 or p120 codingregion sequence could be used, those complementary to the transcribeduntranslated region are most preferred.

The antisense molecules should be delivered to cells which express thep101 regulatory subunit in vivo, e.g., cells of hempatopoetic originsuch as platelet, and neutrophils and other leukocytes. A number ofmethods have been developed for delivering antisense DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue or cell derivation site, or modified antisense molecules,designed to target the desired cells (e.g., antisense linked to peptidesor antibodies that specifically bind receptors or antigens expressed onthe target cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous p101 or p120 transcriptsand thereby prevent translation of the p101 or p120 mRNA. For example, avector can be introduced in vivo such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in mammalian cells. Expression of thesequence encoding the antisense RNA can be by any promoter known in theart to act in mammalian, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include but are not limitedto: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3' long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42), etc. Any type ofplasmid, cosmid, YAC or viral vector can be used to prepare therecombinant DNA construct which can be introduced directly into thetissue or cell derivation site; e.g., the bone marrow. Alternatively,viral vectors can be used which selectively infect the desired tissue orcell type; (e.g., viruses which infect cells of hematopoietic lineage),in which case administration may be accomplished by another route (e.g.,systemically).

Ribozyme molecules designed to catalytically cleave p101 or p120 mRNAtranscripts can also be used to prevent translation of p101 or p120 mRNAand expression of p101 regulatory subunit. (See, e.g., PCT InternationalPublication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990,Science 247:1222-1225). While ribozymes that cleave mRNA at sitespecific recognition sequences can be used to destroy p101 or p120mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5'-UG-3'. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach, 1988, Nature, 334:585-591. There are hundreds of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofhuman p101 or p120 cDNA (FIG. 3). Preferably the ribozyme is engineeredso that the cleavage recognition site is located near the 5' end of thep101 or p120 mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter "Cech-type ribozymes") such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug et al., 1986, Nature,324:429-433; published International Patent Application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in p101 or p120.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the p101 regulatory subunitin vivo, e.g., neutrophils. A preferred method of delivery involvesusing a DNA construct "encoding" the ribozyme under the control of astrong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous p101 or p120 messages and inhibit translation. Becauseribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous p101 or p120 gene expression can also be reduced byinactivating or "knocking out" the p101 or p120 gene or its promoterusing targeted homologous recombination. (E.g, see Smithies et al.,1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512;Thompson et al., 1989 Cell 5:313-321; each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functionalp101 regulatory subunit (or a completely unrelated DNA sequence) flankedby DNA homologous to the endogenous p101 or p120 gene (either the codingregions or regulatory regions of the p101 or p120 gene) can be used,with or without a selectable marker and/or a negative selectable marker,to transfect cells that express p101 regulatory subunit in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the p101 or p120 gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive p101 regulatory subunit (e.g., see Thomas & Capecchi 1987 andThompson 1989, supra). However this approach can be adapted for use inhumans provided the recombinant DNA constructs are directly administeredor targeted to the required site in vivo using appropriate viralvectors.

Alternatively, endogenous p101 or p120 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the p101 or p120 gene (i.e., the p101 or p120 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the p101 or p120 gene in target cells in the body. (See generally,Helene, C. 1991, Anticancer Drug Des., 6(6):569-84; Helene, C. et al.,1992, Ann, N.Y. Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays14(12):807-15).

In yet another embodiment of the invention, the activity of p101regulatory subunit can be reduced using a "dominant negative" approachto interfere with trimeric G protein activation of PI3K. To this end,constructs which encode defective p101 regulatory subunits can be usedin gene therapy approaches to diminish the activity of the p101regulatory subunit in appropriate target cells. For example, nucleotidesequences that direct host cell expression of p101 regulatory subunitsin which the Gβγ interacting domain is deleted or mutated can beintroduced into hematopoietic cells (either by in vivo or ex vivo genetherapy methods described above). Alternatively, nucleotide sequenceswhich encode only a functional domain of p101 could be used as aninhibitor of native p101/p120 interactions. Alternatively, targetedhomologous recombination can be utilized to introduce such deletions ormutations into the subject's endogenous p101 or p120 gene in the bonemarrow. The engineered cells will express non-functional receptors(i.e., a regulatory subunit that is capable of binding the catalyticsubunit, but incapable of stimulating the catalytic activity in responseto G protein activation). Such engineered cells, i.e. neutrophils orother leukocyte lineages, should demonstrate a diminished response toactivation of G protein linked receptors to extracellular chemokines,resulting in reduction of the inflammatory phenotype.

2. RESTORATION OR INCREASE IN p101 REGULATORY SUBUNIT EXPRESSION ORACTIVITY TO PROMOTE IMMUNE SYSTEM ACTIVATION

With respect to an increase in the level of normal p101 or p120 geneexpression and/or p101 regulatory subunit gene product activity, p101 orp120 nucleic acid sequences can be utilized for the treatment ofhematopoietic lineage cell activation disorders, including reducedimmune system responses to chemokines. Where the cause of the immunesystem disfunction is a defective p101 regulatory subunit, treatment canbe administered, for example, in the form of gene replacement therapy.Specifically, one or more copies of a normal p101 gene or a portion ofthe p101 gene that directs the production of a p101 gene productexhibiting normal function, may be inserted into the appropriate cellswithin a patient or animal subject, using vectors which include, but arenot limited to adenovirus, adeno-associated virus, retrovirus and herpesvirus vectors, in addition to other particles that introduce DNA intocells, such as liposomes.

Because the p101 or p120 gene is expressed in the hematopoietic lineagecells, including the neutrophils and other leukocytes, such genereplacement therapy techniques should be capable of delivering p101 orp120 gene sequences to these cell types within patients. Alternatively,targeted homologous recombination can be utilized to correct thedefective endogenous p101 or p120 gene in the appropriate cell type;e.g., bone marrow cells or neutrophils and/or other leukocytes. Inanimals, targeted homologous recombination can be used to correct thedefect in ES cells in order to generate offspring with a correctedtrait.

Finally, compounds identified in the assays described above thatstimulate, enhance, or modify the signal transduced by activated p101regulatory subunit, e.g., by activating downstream signalling proteinsin the p101 regulatory subunit cascade and thereby by-passing thedefective p101 regulatory subunit, can be used to achieve immune systemstimulation. The formulation and mode of administration will depend uponthe physico-chemical properties of the compound.

G. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The compounds that are determined to affect p101 or p120 gene expressionor p101 regulatory subunit activity, or the interaction of p101 with anyof its binding partners including but not limited to the catalyticsubunit, can be administered to a patient at therapeutically effectivedoses to treat or ameliorate hematopoietic cell activation disorders,including inflammatory response disorders such as arthritis, includingrheumatoid arthritis, septic shock, adult respiratory distress syndrome(ARDS), pneumonia, asthma and other lung conditions, allergies,reperfusion injury, atherosclerosis and other cardiovascular diseases,Alzheimer's disease, and cancer. A therapeutically effective dose refersto that amount of the compound sufficient to result in amelioration ofsymptoms of hematopoietic lineage cell activation disorders.

1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

2. FORMULATIONS AND USE

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(eacr, almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of thedisclosure or the protection granted by Letters patent hereon.

VI. EXAMPLE Purification and characterization of porcine Gβγ-activatedPI3K activities

A. MATERIALS & METHODS

1. PI3K ASSAYS

Purified aliquots of Sf9-derived or pig neutrophil cytosol-derived PI3Kwere diluted in ice-cold 0.12M NaCl, 25 mM HEPES, 1 mM EGTA, 1 mM DTT, 1mg-ml⁻¹ BSA, 1% betaine, 0.02%, w/v, Tween-20, pH 7.4, 0° C. to anappropriate extent, then 5 μl aliquots were stored on ice until assayed.If the PI3K assays were performed on immunoprecipitates from U937 cells(see following examples) then the PI3K was immobilized on 10 μl ofpacked protein G Sepharose beads in an ice-cold buffer defined above. 30μl of a mixture of phospholipids with or without Gβγs and/or Gαs (eitherGDP-bound or activated) was added to the 5 μl fractions, or 10 μl ofbeads, and mixed. After 10 minutes on ice, 5-10 μl of last wash buffer,supplemented with MgCl₂ to give a final concentration in the extantassay volume of 3.5 mM, was added and mixed. Six minutes later, 5 μl oflast wash buffer was added (to give a final assay volume of 50 μl)containing y³² P!-ATP (typically 10 μCi assays⁻¹, Amersham, PB10168) and3.5 mM MgCl₂, tubes were mixed and transferred to a 30° C. water bath.Assays were quenched after 15 minute with 500 μl ofchloroform:methanol:H₂ O (29:54:13.1, v/v/v). One μl of 100 mM ATP, 103μl of 2.4 HCl and 434 μl of chloroform were subsequently added togenerate a two phase `Folch` solvent distribution. After mixing andcentrifugation, the lower phases were removed and transferred to cleantubes containing 424 μl of fresh `upper phase` (methanol:1M HCl:chloroform; 48:47:3, v/v/v). After mixing and centrifugation the lowerphase was removed to a fresh tube (during the purification of porcineneutrophil PI3Ks, ³² P!-lipid product was quantitated at this point witha geiger counter), dried under vacuum, deacylated and resolved by TLC onPEI plates (with 0.6M HCL; PtdIns(3,4,5)P₃ has an Rf of 0.47) (Stephenset al., 1994, Cell 77:83-93). In some experiments the dried lipid wasredissolved in chloroform: methanol/2:1, v/v), applied to a potassiumoxalate-impregnated TLC plated and resolved in a mixture of chloroform:acetone: methanol: acetic acid: H₂ O (40: 15: 13: 12: 8, v/v/v/v/v;Traynor-Kaplan et al., 1988).

The lipid/G protein subunit mixtures were prepared as follows:PtdIns(4,5)P₂ (which was prepared from Folch phosphoinositide fractionsby 2 cycles of chromatography on immobilized neomycin) and PtdEtn(Sigma) were dried under vacuum (sufficient to give final concentrationsin the assay of 50 μM and 0.5 mM, respectively). In some experimentsPtdIns4P (prepared similarly to the PtdIns(4,5)P₂) and/or PtdIns (Sigma)were included. The dried lipid was bath-sonicated (at room temperature)into final wash buffer (above) supplemented with 0.1%, sodium cholate.After cooling on ice, portions of this dispersed lipid stock were mixedwith a mixture, totaling 1 μl assay⁻¹, of Gβγ storage buffer (1%cholate; 50 mM HEPES, pH 7.5, 4° C., 0.1M NaCl; 1 mM DTT; 0.5 mM EDTA),active Gβγ, or an equivalent volume of boiled Gβγ from 3-7 mg-ml⁻¹stocks in storage buffer. In some experiments the 1 μl would include Gαsubunits or their storage buffer in which case the Gβγs were premixedwith the Gα subunits (either GDP bound or activated; see below) for 10minutes on ice.

Gα subunits (an equimolar mixture of Gα:o, i, i₂ and i₃ prepared asdescribed in, and stored in the same buffer as the Gβγ subunits exceptsupplemented with 10 μM GDP) were activated by incubation on ice with 10mM NaF, 30 μM AlCl₃ (A/F) for 10 mins; assays into which these subunitswere diluted also contained A/F.

a) Protein Purification

Pigs blood (42 batches) was collected directly into anti-coagulant in 21containers. Within 40 minute of collection the blood/anti-coagulant wasmixed with 3% (w/v) polyvinylpyrrolidone (PVP, 360 kD) in isotonicsaline (4.2 of blood mixture: 0.8 of PVP). After 35 minutes standing (in25 containers) the erythrocytes had settled adequately to allow thesupernatant to be siphoned off and centrifuged (8 minutes, 1000×g) in 1liter containers. Approximately 28 liters of this primary supernatantwas recovered. The sedimented cells were resuspended in Hank's salinesuch that the final accumulated volume was contained in two 1 litercentrifuge bottles. The cells were sedimented by centrifugation (8minute 1000×g). Supernatants were aspirated and the cell pellet washypotonically shocked (to lyse any residual, contaminating erythrocytes)by the addition of 70 mls of ice-cold H₂ O. After 25-30 seconds ofmixing, 77 mls of 10×Hank's saline (without calcium) was added. Afterone wash with Hank's saline, the cells were combined into one centrifugebottle, pelleted and resuspended in 500 mls of ice-cold; 40 mM TRIS, pH7.5, 4° C.; 0.12M NaCl;

2.5 mM MgCl₂. Di-isopropylfluorophosphate was added (final concentration0.5 mM), after 5 minute on ice the cells were pelleted (approximately80-90 mls packed volume) and resuspended in 300 mls of ice-cold 40 mMTRIS, pH 7.5; 0.1M NaCl; 2.5 mM MgCl₂ ; 1 mM EGTA; 0.2 mM EDTA andantiproteases I. The cell suspension was sonicated (Heat Systems Probesonicator, setting 9.25, 4×15 seconds with 1 minute mixing, on ice,between each burst) then centrifuged (2000×g 10 min) to sedimentunbroken cells and nuclei (less than 5% of cells remained intact). Thesupernatant was centrifuged (at 100,000×g 60 min, 4° C.) and thesupernatants were decanted, pooled, mixed with EDTA, betaine and DTT(final concentrations of 5 mM, 1% and 1 mM; respectively) and finallyfrozen in liquid nitrogen and stored at -80° C. Cytosolic fractionsprepared in this manner typically had a protein concentration of 8 to 9mg-ml⁻¹ (about 2.5 g protein per preparation). Once the cytosol from theequivalent of 750 liters of blood (18 to 42 preparations, 9×10¹² cells,40 g protein) had been collected and stored at -80° C., they were thawedin three batches separated by 5 hr. From this point onwards allprocedures were carried out at 2°-4° C.

The freshly thawed cytosol was supplemented with Tween-20 (0.05%, w/v,final concentration), centrifuged (20,000×g for 30 min, 2° C.) filtered(5 μm cellulose nitrate, 4.5 cms diameter; Whatman) dilutedapproximately 2.5× with buffer K (see below) to a final conductivity of200 μS (at 4° C.), then loaded (12.5 ml-min⁻¹ with a peristaltic pump)onto a 800 ml (5 cms diameter) column of fast flow Q Sepharoseequilibrated with buffer K. The total volume of diluted cytosol wasapproximately 15.5 liters. Once loaded, the column was washed with 1liter of buffer K then eluted with a 4.5 liter, linear gradient, of 0.1to 0.6M NaCl in buffer K at 8 ml-min⁻¹. Fifty 1 ml fractions werecollected. The conductivity and Absorbance (at 280 nm) of the columneluate were monitored continuously (and in all subsequent steps). BufferK had the following composition: 40 mM TRIS/Hl, 1 mM EGTA, 0.2 mM EDTA,1% betaine; 0.05% w/v Tween 20, 5 mM β-glycerophosphate pH 7.5 at 4° C.,15 mM β-mercaptoethanol with 4 μgml⁻¹ each of antipain, leupeptin,bestatin, pepstatin A and aprotinin and 0.1 mM PMSF (`antiproteasesII`). This solution, as well as those that follow, was 0.2 μm filtered.

Once the relevant fractions had been identified by PI3K assays, theywere pooled and immediately loaded (10 ml-min⁻¹) onto a 1.8 1 column (5cms diameter) of Sephadex-G25-fine, which had been pre-equilibrated with18 1 buffer L (only last 2 liters with antiproteases II), (buffer Lcontained: 5 mM β-glycerophosphate, 20 mM KCl, 0.05% w/v Tween 20, 1%betaine, 0.1 mM EDTA, 10 mM potassium phosphate pH 7.0 at 4° C., 15 mMβ-mercaptoethanol plus antiproteases II). The desalted pool from Qsepharose was immediately loaded (5 ml-min-⁻¹) onto 80 ml ofhydroxylapatite (2.6 cms diameter; Macroprep-ceramic, BioRad)equilibrated with 1 liter of buffer M (5 mM β-glycerophosphate; 10 mMpotassium phosphate, pH 7.0, 4° C., 0.05% w/v Tween 20, 1% Betaine, 15mM β-mercaptoethanol) at a flow rate of 10 ml-min⁻¹, and then with 100mls of buffer N, (comprised of buffer M supplemented with 0.1 mM EDTAand antiproteases I) immediately prior to loading the sample. Afterloading, the column was washed with 100 mls of buffer N and eluted withan 100 ml linear gradient of 0.05 to 0.35M potassium phosphate in bufferN (4 ml-min⁻¹). 25 ml fractions were collected and assayed forGβγ-stimulated PI3K activity.

Relevant fractions were pooled (typically a total of 100 mls), diluted3× with buffer 0 (to a conductivity of 250 μS, 4° C.) and loaded (1.1ml-min⁻¹) onto Heparin Sepharose HR (1.6 cms diameter column that hadbeen pre-equilibrated with 150 mls of buffer O (see below, at 2ml-min⁻¹). After loading, the column was washed with buffer O (30 mls)and eluted with a 140 ml linear gradient of 0.1-0.7M KCl in buffer O(flow rate 1 ml-min⁻¹), the elute was collected in 5 ml fractions.Buffer O was: 20 mM HEPES, 1 mM EGTA, 0.2 EDTA, 0.05% w/v/Tween 20, 1.0%butane, 1 mM β-glycerophosphate pH 7.2, 4° C., 15 MM β-mercaptoethanol,plus antiproteases II.

Gβγ-stimulated PI3K activity eluted from Heparin sepharose HR in twopeaks, designated peaks A and B. Both A and B were further purified bysequential use of the same combination of columns. Peak A was in 15 mls(0.4M KCl) and was diluted 8 fold into buffer P (see below) to a finalconductivity of 200 μS, 4° C.), peak B was in 15 mls (0.6M KCl) and wasdiluted 10× into buffer P (to a final conductivity of 200 μS, 4° C.).Dilution was immediately prior to loading at 1 ml-min-⁻¹ onto a Mono Q5/5 HR column pre-equilibrated with 20 mls of buffer P. After loading,the column was washed with 5 mls of buffer P. Eluate was collected in0.5 ml fractions. Buffer P contained: 10 mM Tris, 1 mM EGTA, 0.2 EDTA,0.05% w/v Tween 20, 1% betaine, 1 mM β-glycerophosphate, pH 7.1, 4° C.,15 mM β-mercaptoethanol plus antiproteases II.

The relevant fractions from Mono Q (A) and (B) were pooled independently(both had a total volume of 3 mls) concentrated with an ultrafiltrationunit (50 kD cut-off prewashed with buffer P) to 0.8 mls, centrifuged(10,000×g for minutes, 0° C.) and loaded (0.25 ml-min⁻¹) directly onto ahigh performance size exclusion chromatography column (V_(o) 72 mls,V_(t) 172 mls) pre-equilibrated with buffer Q (see below; 2 literswithout antiproteases II, then 150 mls with antiproteases II) 1.5 mlfractions were collected just prior to the V_(o). Buffer Q contained:0.17M KCl, 1% betaine, 0.05% w/v Tween 20, 1 mM β-glycerophosphate, 1 mMEGTA, 0.2 mM EDTA, 1.5 mM potassium phosphate, 40 mM HEPES, pH 6.9 at 4°C., 15 mM β-mercaptoethanol.

Relevant fractions from A and B were pooled independently (both had atotal volume of 6 mls) diluted with buffer R to 24 mls (finalconductivity 250 μS, 4° C.) and loaded (0.8 ml-min⁻¹) onto anacrylic-based cation-exchange HPLC column (2.5 mls volume, BioRad) andeluted with a 25 ml linear gradient of KCl (0.1 to 0.6M) in buffer R.The eluate was collected in 1 ml fractions. Buffer R contained: 1%betaine, 0.05% w/v Tween 20, 1 mM EGTA, 0.2 mM EDTA, 20 mM HEPES, pH 6.8at 4° C., 15 mM β-mercaptoethanol plus antiproteases II.

Relevant fractions were pooled (3 mls for (A), 2 mls for (B)), diluted7× with buffer S (final conductivity of 180 μS, 4° C.) and loaded (0.15ml-min⁻¹); onto a Mini Q column (0.24 mls, operated on a PharmaciaSmart™ system). The column was washed with 1 ml of buffer S and elutedwith a linear gradient of NaCl (0.1 to 0.5M NaCl) in buffer S at 0.1ml-min-⁻¹. The elute was collected 75 μl fractions. Buffer S contained:1% betaine, 0.05% w/v Tween 20, 1 mM EGTA, 0.2 mM EDTA, 2 mMβ-glycerophosphate, 10 mM TRIS, pH 7.7, 4° C., 1 mM DTT (withoutantiproteases).

Protein concentrations throughout the purification were was estimated infour ways: (a) with a protein binding dye (BioRad; this was only used onlysates, cytosolic and Q sepharose fractions); (b) by integration of Abs280 nm peak areas (this was calibrated by using the dye binding assay);(c) proteins on filters were stained with Ponceau S and compared withthe staining intensity of defined aliquots of a similarly immobilizedstandard; and (d) proteins resolved by SDS-PAGE and stained withCoomassie R250 were compared with aliquots of proteins of knownconcentration run on the same gel.

Final preparation of PI3K (or first stage purified material) wereincubated with 100 nM ³ H!-17-hydroxy-wortmannin (17.7 Ci mmol⁻¹,Amersham, custom made), resolved by SDS-PAGE, stained with CoomassieBlue, and photographed. ³ H! was then detected fluorographically.

VII. RESULTS

Analysis of porcine neutrophil cytosol by an ion-exchange chromatographyshowed it contained a Gβγ-activated PI3K activity of similar propertiesto ones already described in U937 and osteosarcoma cells. Use of ³H!-17-hydroxy-wortmannin as a probe identified a doublet of proteins ofapparent size 117 kD and 120 kD which eluted in the fractions containingGβγ-activated PI3K activity, and further, that they were at 2-4% of thelevels of ³ H!-17-hydroxy-wortmannin bound by p110α and/or p110γ (seeFIGS. 5). This peak of Gβγ activated PI3K activity was purified further(all figures and tables detail the purification of the preparations ofPI3Ks that were ultimately sequenced). During this procedure, it splitinto two peaks (A and B) both which displayed apparent, native,relative, molecular masses of 220 kD. Once essentially pure, as assessedby Coomassie-stained SDS-PAGE gels, it was clear that both activitiesco-migrated with two proteins: (A) with proteins of 117 kD (whichspecifically bound ³ H!-17-hydroxy-wortmannin and was assumed thereforeto be the catalytic subunit) and 101 kD; and (B) with proteins of 120 kD(which also bound ³ H!-17-hydroxy-wortmannin) and 101 kD. This resultindicated that the PI3K activities were p117/p101 and 120/p101heterodimers in their native state. In their final forms PI3Ks A and Bhad been purified approximately 180,000× and 380,000× from neutrophillysates (1,000,000,000× from blood) with 5.5% overall recovery ofactivity. Table 1 defines the recoveries of protein and PI3K activitythrough each step).

                                      TABLE 1    __________________________________________________________________________    Purification of pig leukocyte G-protein βγ subunit activated    PI3K's                Total          Total                protein                       Pool    Activity                                   Fold    Pool of activity                in pools                       Volume  in pools                                   Purification    __________________________________________________________________________    Cytosol     40  g  151     100%                                     1    Q-Sepharose 1.5 g  40  mls 90%  24    (desalted)  (1.5 g)                       (450 mls)                               (124%)    Hydroxylapatite                162 mg 100 mls 125%                                    309    Heparin Peak A                19  mg 15  mls 46%  970    Sepharose            Peak B                12  mg 15  mls 53% 1769    Mono Q pool            A   5.4 mg 3   mls 16% 1187            B   1.4 mg 3   mls 15% 4291    Size exclusion            A   0.722                    mg 6   mls 16% 8902    (850 μl applied)            B   0.2 mg 6   mls 15.5%                                   51038    Cation  A   0.13                    mg 3   mls  6% 18489    Exchange            B   0.014                    mg 2   mls 9.5%                                   271761    Mini Q pool            from A                15  μg                       0.225                           mls 2.2%                                   58754            from B                10  μg                       0.225                           mls 3.1%                                   174151    __________________________________________________________________________

These extents of enrichment are consistent with the quantities of ³H!-17-hydroxy-wortmannin bound by these proteins compared to p85/p110family members. All of these proteins are thus considered to be of lowabundance.

Purified preparations of PI3Ks A and B were indistinguishable on thebasis of their lipid kinase activities. Both preparations were (a)activated over 100× by Gβγ subunits, in a Gα-GDP-sensitive fashion, (b)completely inhibited by 100 nM wortmannin (with 5 μM ATP in the assays),(c) insensitive, either in the presence or absence of Gβγs, to tyrosinephosphorylated peptides which activate p85/p110 family PI3Ks five fold(see FIG. 6), and (d) able to 3-phosphorylate PtdIns, PtdIns4P andPtdIns(4,5)P₂ (the identity of the products was established bysequential deacylation and deglyceration followed by anion-exchange HPLCanalysis of the ³² P!-labelled water-soluble products with internal ³H!-labeled standards). Further, the purified preparations of PI3K A andB displayed the lowest apparent Km for the latter substrate (8 and 10 μMfor A and B, respectively) utilizing the γ-phosphate of ATP as thephosphate donor (results not shown).

VIII. EXAMPLE PEPTIDE SEOUENCING OF PORCINE Gβγ-ACTIVATED PI3K A AND B

In this example, both porcine PI3K proteins were analyzed by amino acidsequencing. PI3Ks A and B, purified from the equivalent of 40 g ofcytosolic protein, were Western blotted onto nitrocellulose, stainedwith Ponceau S, the bands corresponding to all four subunits wereexcised, treated with trypsin and processed for internal amino acidsequence analysis.

A. MATERIALS AND METHODS

Generation of peptides and peptide sequencing. Aliquots of protein forsequencing were Western blotted (in a wet blotter) onto nitrocellulose(0.45 μm pore size BA85; Schleicher and Schuell). The transfer buffercontained 192 mM glycine, 25 mM TRIS and 10% v/v methanol. Prior toassembling the final transfer unit the Whatman No. 1 filter papersupports on the (-) side of gel/filter sandwich, and the gel (1 mmthick), were soaked (2-3 mins) in transfer buffer containing 0.0005w(w/v) SDS. The transfer was for 16 h at a fixed 35 V (0.25 to 0.35 Amps,at 5° C.). The filters were stained with 0.1% Ponceau S in 1% aceticacid for 1 min, then destained for 1 minute in 1% acetic acid.Approximately 85-90% of the protein loaded on the gel was recovered thefilter. The bands of interest were excised from the nitrocellulose andprocessed for internal amino acid sequence analysis as described (Tempstet al., 1990, Electrophoresis 11:537-552), with modifications (Lui etal., 1996). Briefly, in situ proteolytic cleavage was done using 0.5 μgtrypsin (Promega, Madison, Wis.) in 25 μl 100 mM NH₄ HCO₃ (supplementedwith 1% Zwittergent 3-16) at 37° C. for 2 hours. The resulting peptidemixture was reduced and S-alkylated with, respectively 0.1%β-mercaptoethanol (BioRad, Richmond, Calif.) and 0.3% 4-vinyl pyridine(Aldrich, Milwaukee, Wis.), and fractionated by reversed phase HPLC. Anenzyme blank was done on an equally sized strip of nitrocellulose.

HPLC solvents and system configuration were as described (Elicone etal., 1994), except that an 2.1 mm 214 TP54 Vydac C4 (Separations Group,Hesperia, Calif.) column was used with gradient elution at a flow rateof 100 μl/min.

Identification of Trp-containing peptides was done by manual ratioanalysis of absorbances at 297 and 277 nm, monitored in real time usingan Applied Biosystems (Foster City, Calif.) model 1000S diodarraydetector (Erdjument-Bromage et al., 1994). Fractions were collected byhand, kept on ice for the duration of the run and then stored at -70° C.before analysis.

Purified peptides were analyzed by combination of automated Edmandegradation and matric-assisted laser-desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry; details about thiscombined approach, including mass-aided post-chemical sequencingroutines can be found elsewhere (Geromanos et al., 1994, Techniques inProtein Chemistry V 143-150; Elicone et al., 1994, J. Chromatogr.676:121-137; Erdjument-Bromage et al., 1994, Protein Sci. 3:2435-2446).After storage, column fractions were supplemented with neat TPA (to givea final concentration of 10%) before loading onto the sequencer disksand mass spectrometer target. Mass analysis (on 2% aliquots) was carriedout using a model Voyager RP MALDI-TOF instrument (Vestec/PerSeptive,Framingham, Mass.) in the linear mode, with a 337 nm output nitrogenlaser, an 1.3 m flight tube and α-cyano-4-hydroxy cinnamic acid (premadesolution obtained from Linear Sci., Reno, Nev.) as the matrix. A 30 kVion acceleration voltage (grid voltage at 70%, guide wire voltage at0.1%) and -2.0 kV multiplier voltage were used. Laser fluence and numberof acquisitions were adjusted as judged from optimal deflections ofspecific maxima, using a TDS 520 Tektronix (Beaverton, Oreg.) digitizingoscilloscope. Mz (mass to charge) spectra were generated from thetime-of-flight files using GRAMS (Galactic Ind., Salem, N.H.) dataanalysis software. Every sample was analyzed twice, in the presence andabsence of two calibrants (25 femtomoles each of APID and p8930), asdescribed (Geromanos et al., 1994). Chemical sequencing (on 95% of thesample) was done using a model 477A instrument from Applied Biosystems(AB). Stepwise liberated PTH-amino acids were identified using an`on-line` 120A HPLC system (AB) equipped with a PTH C18 (2/1×220 mm; 5micron particle size) column (AB). Instruments and procedures wereoptimized for femtomole level phenyl thihydantoin amino acid analysis asdescribed (Erdjument-Bromage et al., 1994, Protein Sci. 3:2435-2446;Tempst et al., 1994, Methods CompanionMeth. Enzymol. 6:284-261).

Peptide average isotopic masses were summed from the identified residues(including the presumed ones) using ProComp version 1.2 software(obtained from Dr. P. C. Andrews, University of Michigan, Ann Arbor,Mich.).

A doubly tyrosine phosphorylated peptide (18 residues) based on thesequence surrounding tyrosines 740 and 751 in the PDGF βR was preparedby the microchemical facility at the Babraham Institute.

B. RESULTS

Peptide sequence data immediately resolved several issues regarding therelationships between these proteins. The p101 s derived from both PI3KsA and B were identical and further a relatively common allelic-variantwas identified at 483 in the ORF, (marked in FIG. 4) such that a serinewas replaced by a glycine. p117 and p120 displayed virtually identicaltryptic HPLC profiles and all apparently common peptides that weresequenced from both species were identical with the exception of aamino-terminal blocked peptide from p117 (see below). Peptide sequenceinformation was then used to design probes for retrieving the nucleotidesequence encoding these proteins.

IX. EXAMPLE Cloning of the cDNAs encoding porcine p120 and p101

Degenerate oligonucleotide probes, based on the sequence of a peptidefrom porcine p120 and a peptide from porcine p101 were used to screen anoligo-dT-primed, amplified, cDNA library (made from pig neutrophil polyA-selected RNA). Described blow is the cloning and characterization ofcDNA's encoding both the p101 and p120 proteins.

A. MATERIALS AND METHODS

We prepared 0.7 mg total RNA from 4.2×10⁹ pig neutrophils (Chomczynski &Sacchi, 1987, Anal. Biochemistry 162:156-159). This RNA was used byStratagene (San Diego, Calif.) to produce PolyA-selected mRNA from whichthey prepared oligodT- and random-primed cDNA libraries in λZAPII(approximately 5.4×10⁻⁶ and 3.2×10⁶ primary p.f.u. respectively).Amplified libraries were constructed from approximately 2×10⁶ originalrecombinants and these were used to screen for p120 and p101 cDNAs bystandard procedures.

2.5×10⁶ plaques derived from the oligodT-primed library were screenedusing a 32P!-labelled oligo based on peptide sequence from p120 CA(T/C)GA(T/C) TT(T/C) ACI CA(A/G) CA(A/G) GTI CA(A/G) GTI AT(T/C/A) GA(T/C)ATG! (SEQ ID NO:5). Twelve positive clones were identified isolated asBluescript™ based plasmids and both DBA strands sequenced (using the ABIautomatic sequencing facility at the Babraham Institute). The inserts ofthese plasmids represented a series of overlapping clones with twoclones defining a full length ORF encoding all of the peptide sequencederived from p117/p120 tryptic digests (FIG. 4).

0.9×10⁶ plaques derived from the oligodT-primed library were screenedusing a ³² P!-labelled oligo based on peptide sequence from p101GCITA(T/C)ATGGA(A/G)GA(T/C)ATIGA(A/G)GA! (SEQ ID NO:6). 1 positive clonewas identified, isolated and sequenced. The 5' end of this clone (D11)represented part of the sequence for one of the tryptic peptides, thusidentifying it as a partial clone. A further 0.6×10⁶ plaques from theoligoT-primed library were screened using a ³² P!-labelled Cla-1restriction fragment derived from D11. Sixty-six positive clones wereidentified, 49 of which were isolated and some partially sequenced.These represented a series of overlapping clones all of which containedD11 sequence but all of which were smaller than D11 itself. 3.5×10⁶plaques from the random-primed library were then screened using a ³²P!-labelled Apa-1 restriction fragment derived from D11. Ninety-eightpositive clones were identified. These clones were re-screened (at thestage of primary plaque isolates) by a PCR-based approach using primersdesigned against the Bluescript™ vector (either `forward` and `reverse`primers) and internal D11 sequence. This enabled us to identify(independent of orientation) the longest potential N-terminal extensionsencoding p101 sequence. The 3 clones giving the largest PCR-fragmentwere isolated and sequenced. These represented overlapping clones which,together with D11, defined a full length ORF encoding all of the peptidesequence derived from the p101 tryptic digest (FIG. 1 and FIG. 2)

B. RESULTS

Two clones defined a full length ORF encoding all of the p117/p120tryptic peptides (see FIGS. 1-4). Of three potential start methionines,the central one was identified as active (in contrast to the assumptionof Stoyanov et al., 1995, Science 269:690-693) on the basis of theprecise match between the measured mass of an amino-terminal blocked120-derived peptide and the theoretical masses of amino-terminalpeptides that would be derived as a result of initiating translation ateach of the three methionines. As such, p120 has a theoretical size of126 kD. Comparison of the mass of the amino-terminal blocked peptideproduced from p117 with the relevant regions of the amino-terminal endof p120 indicates no precise matches (allowing for usual amino-terminalblocking). Hence p117 is unlikely to be a proteolytically orpost-translationally modified form of p120, nor is it likely to resultfrom use of a second translation start point within the p120 message.However, a cDNA with an ORF encoding p117 has not been isolated.

The protein and DNA sequences defining p120 were used to search databases for similar structures. Similarities with all previously clonedPI3Ks were identified. However, the sequence was nearly identical,allowing for species differences, to p110γ (Stoyanov et al., 1995). Theonly significant discrepancy between our sequence and that Stoyanov etal., is found in the extreme COOH terminus. On the basis of primarystructure only, the identification of a COOH terminal pleckstrinhomology domain in p120 could not be confirmed.

By utilizing several overlapping fragments, derived from both oligo-dTand random-primed, pig neutrophil-derived, cDNA libraries, a full lengthORF encoding all of the peptide sequence derived from p101 has beendefined. A p101 -derived, amino-terminal blocked peptide was identified;its mass was precisely equivalent to that predicted for anamino-terminal acetylated version of the first 12 residues defined bythe predicted start in the ORF described. The predicted relativemolecular mass of p101 is 97 kD. Although the protein and DNA sequencedata bases were searched for similar structures or sub-structures, nosignificant matches were identified.

X. EXAMPLE Cloning of the cDNAs encoding human p120 and p101

Radiolabeled PCR products derived from the porcine p101 and p120 cDNAsequences were successfully used to screen human cDNA libraries fortheir human homologs. The cloning and characterization of cDNA'sencoding human p101 and p120 is described below.

A. Human p101

A human monocyte cDNA library (Clontech, Palo Alto, Calif.; mRNA source:U937 cell line, PMA treated at 50 ng/ml for 3.5 days) was screened witha radiolabeled PCR product corresponding to bp 887-1287 of the porcinep101 cDNA sequence. λgt11 phages containing the human cDNA library wereplated, as described in the Clontech Lambda Library Protocol Handbook,and transferred to nylon filters. Filter-bound DNA was denatured byautoclaving the filters for one minute, after which the filters wereprehybridized for 2 hours at 42° C. in 50% formaldehyde, 5× Denhardts,5× SSC, 0.05 mg/ml salmon sperm DNA, 0.05M NaPO₄ at pH6.8, 1 mM sodiumpyrophosphate and 1% SDS. The radiolabeled PCR probe was denatured byboiling for 5 minutes, cooled on ice for 15 minutes, and then added tothe filters in the hybridization buffer at a final concentration of 1million cpm/ml. Hybridization proceeded overnight at 42° C. withconstant agitation. Filters were then washed once in 2× SSC, 1% SDS atroom temperature for 20 minutes, followed by a wash in 2× SSC, 1% SDS at42° C. for twenty minutes, and finally a wash at 0.2× SSC, 1% SDS at 42° C. for 20 minutes. After autoradiography, positive clones wereisolated according to the Clontech Library Handbook by picking the areaaround and including the positive clone and eluting the phage into PhageDilution Buffer. The phages were then replated at a density allowing forisolation of a single phage plaques, grown overnight, and transferred tonylon filters. The filters were hybridized with the same radioactive PCRprobe as above, washed, and subjected to autoradiography. Single, pure,positive phage plaques were picked, the phage eluted into Phage DilutionBuffer and then replated to give a confluent lawn. The plaque purifiedphage lawn was eluted into Phage Buffer and lambda phage DNA isolatedaccording to the protocol in the Clontech Library Protocol Handbook.

Purified lambda DNA was cut with the restriction enzyme EcoRI, separatedon an agarose gel, and the EcoRI DNA insert isolated and recloned intoEcoRI linearized Bluescript KS+. Bluescript plasmids containing theseDNA inserts were then sequenced. Five of the independently isolated cDNAclones contained sequences homologous to the porcine p101 cDNA sequencebetween nucleotide 1137 and the stop codon at nucleotide 3021. Relativeto the porcine p101 sequence, the isolated human clones contained anadditional 544 bp of 3' untranslated sequence.

Because the porcine sequence is very GC rich at the 5' end, theprobability of finding a full length clone in an oligo-dT primed libraryis low. Therefore, we decided to use a library that was both oligo dTand random primed in order to increase the probability of finding aclone that contained the GC rich 5' end. Using the same radiolabeled PCRproduct of the porcine p101 sequence and the same methods describedabove, we screened a human leukocyte 5'-Stretch Plus cDNA Library(Clontech; mRNA source: normal peripheral blood leukocytes) and isolatedseveral clones, one of which contained the 5' end of the human p101sequence. This clone included roughly 300 bp upstream of the codingsequence and the first 1612 bp of the coding sequence. A full lengthcDNA clone containing the entire coding region of the human p101sequence was then constructed by fusing the 5' end of the leukocyteclone (from a HindIII site 77 bp upstream of the start codon to a SacIsite at position 1262) with the monocyte clone (from the SacI site to anXbaI site at position 3014, 305 bp downstream of the stop codon). Theentire sequence of the full-length cDNA clone was determined, and thecoded amino acid sequence deduced.

Since this full length clone was derived from two different clones fromtwo different libraries, the existence of the 3' end of the p101 clonein the leukocyte library was verified. Using five different sets of PCRprimers, we demonstrated that the 3' end of p110, which was cloned fromthe monocyte library, was present in both the monocyte and the leukocytelibraries.

B. Human p120

The human homolog of porcine p120 was cloned by screening a leukocytecDNA library (Clontech; mRNA source: normal peripheral blood leukocytes)with a radioactively labeled PCR fragment of the porcine sequence frombp 875 to 1315, according to the protocol described above for thecloning of human p101. We independently isolated two clones containingthe 5' end of human p120, corresponding to bp 6 to 2414 in the porcinesequence as reported by Stephens et al. The human sequence contains anEcoRI site at bp 2408, and since the cDNA library had been constructedwith EcoRI linkers, the full length human sequence had to be containedin two separate clones, one containing the 5' EcoRI fragment, the othercontaining the 3' EcoRI fragment. The library was therefore rescreenedunder the same conditions as above with a radioactively labelled PCRfragment from the 3' end (bp 2801 to 3395) of the porcine p120 sequence.We independently isolated two clones containing the 3' end of thesequence from position 2408 (human sequence) to the beginning of thepoly A tail at position 5128. The DNA inserts of the two phagescontaining the 5' and the 3' EcoRI fragment were isolated and fused atthe EcoRI site to give a full length cDNA clone (1-5128) containing thetotal coding region (84 to 3390) of the human p120 homolog. The entiresequence of the full-length cDNA clone was determined, and the codedamino acid sequence deduced.

XI. EXAMPLE Expression of porcine p120 and p101 in insect cells

Recombinant, clonal, baculo-viruses (rbv) harboring either aamino-terminal, 6× HIS-tagged p120 (pAcHLT 120) cDNA or aamino-terminal, (EE)-tagged p101 cDNA (pAcO-Gl) p101) were prepared andused to drive production of the above proteins in insect (Sf9) cells.

A. MATERIALS AND METHODS

1. CONSTRUCTION OF EXPRESSION VECTORS

The use of N-terminal PCR and appropriate restriction sites allowed theporcine p120 and p101 ORFs to be manipulated into a form where theycould be inserted in frame into various expression vectors. In eachcase, the first amino acid encoded after the N-terminal tag was thestart methionine. The 3'-untranslated region of the p120 cDNA was usedin full and that of the p101 cDNA truncated at a BamHI site (nucleotide192, FIG. 1). The vectors used for baculovirus-driven expression in Sf9cells were pAcHLT (which encodes an N-terminal `6× His-tag` followed bya thrombin cleavage site, Pharminogen; the p120 ORF was inserted intothe XhoI-EcoRI sites) and pAcO-Gl (which encodes an N-terminal `EE-tag`,ONYX Pharmaceuticals; the p101 ORF was inserted into the EcoRI-NotIsites). All vectors were N-terminally sequenced before use.

2. Sf9 cell transfections and production of recombinant proteins

Sf9 cells were grown in TNM FH with 11% HI-FBS in a spinner flask andwere maintained at between 0.5 and 2×10⁶ cells ml⁻¹. Sf9 cells weretransfected with Insectin™ (Invitrogen) liposomes with linearizedbaculo-gold DNA (Pharminogen) and relevant baculo-virus transfer vectorsas recommended (Invitrogen). The resulting recombinant baculo viruseswere plaque-purified and amplified to yield high-titre viral stocks. Theoptimal (for production of protein) dilutions were determined for eachhigh-titre stock. pAcO-Gl p101 virus were allowed to infect adherent Sf9cells for 2.2 days at 27° C.; pAcHLT p120 virus were allowed to infectin a spinner culture (usually) for 1.9 days at 27° C. Cells wereharvested into ice-cold 0.41% KCl; 2.66% sucrose; 20 mM MgCl₂ ; 8 mMNaH₂ PO₄, pH 6.2, 25° C.; treated with 1 mM di-isopropylfluorophosphate(5 minutes on ice), and washed once in the harvesting solution.Centrifugally packed aliquots of cells were frozen in liquid N₂ andstored at -80° C.

3. Purification of Sf9-derived proteins

Porcine p120 was purified using a metal-ion chelation column (Talon,Clontech). Cell pellets were thawed into 0.10M NaCl; 50 mM sodiumphosphate, pH 8.0, 4° C.; 10 mM Tris.HCl, pH 8.0, 4° C.; 1 mM MgCl₂ andantiproteases I (see above) at a ratio of 1 liter of infected Sf9 cellculture into 50 mls of sonication buffer, probe-sonicated (4×15 secondbursts on ice), and centrifuged (120,000×g for 40 minutes, 4° C.). Thesupernatant was removed and pooled (cloudy supernatant at the top of thetube was removed separately, 0.45 μM filtered with a low-protein bindingfilter and then pooled with the remainder). The cytosolic fraction wassupplemented with Tween-20 and betaine (0.05%, w/v, and 1%,respectively) then pumped onto a column of Talon resin equilibrated inequivalent buffer (1.2 mls of resin per liter of original infected Sf9culture at a linear flow velocity of 20 cms hr⁻¹). This, and allsubsequent steps were carried out at 4° C. The column was sequentiallywashed (same flow) with 20 column volumes each of: Buffer A, 50 mMsodium phosphate, pH 8.0, 4° C.; 10 mM Tris/HCl, pH 8.0, 4° C.; 0.15MNaCl; 1% betaine; 0.05%, w/v, Tween-20; buffer B, 1%, w/v, Triton X-100;0.15M NaCl; 50 mM sodium phosphate, pH 8.0, 4° C.; 10 mM Tris, pH 8.0,4° C.; 1% betaine 0.05%, w/v, Tween-20; buffer C, 0.15M NaCl; 50 mMsodium phosphate, pH 7.1, 4° C.; 1betaine; 0.05%, w/v, Tween-20; bufferD; 0.15M NaCl; 30 mM Tris, pH 7.5, 4° C., 1% betaine, 0.02%, w/v,Tween-20; 10%, v/v, ethylene glycol; 1 mM MgCl₂ ; and then 8 columnvolumes buffer E, comprised of buffer D supplemented with 10MM imidazole(pH 7.5). During the Buffer E wash, 2 ml fractions were collected.Finally, at half the flow used previously, the column was washed withbuffer F, which was comprised of buffer D supplemented with 70 mMimidazole (pH 7.5; final concentration). Typically 1 ml fractions werecollected. With experience fractions were pooled on the basis of the Abs280 nm trace (continuously recorded) and supplemented with 1 mM DTT and1 mM EGTA (final concentrations) immediately. Typically this processyielded 4 mg of p120 per liter of Sf9 culture. The p120 prepared in thismanner was usually greater than 90% pure. The final pool of p120 wasdesalted via a 15 ml column of G-25 superfine equilibrated in buffer G,which was comprised of buffer D supplemented with 1 mM DTT and 1 mM EGTA(final concentrations). `p120 blank` preparations used in someexperiments were prepared in precise parallel to a normal p120preparations except the starting cells were either infected with wildtype baculo-virus, or were uninfected. The final fractions derived fromthese `blank` preparations were pooled on a `parallel volume` basisbecause they contained virtually no protein.

The p120 6× HIS tag contained a thrombin cleavage recognition motif.Careful titration with thrombin and analysis with 6% polyacrylamideSDS-gels revealed two thrombin sites, with similar sensitivities tothrombin, both close to the amino-terminal (because an αCOOH-terminalantibody still immunoprecipitated the twice-cut 120). One site was atthe expected location for the site engineered into the tag; the othersite was approximately 40-residues in from the amino-terminal (in aregion with no favored thrombin recognition sequences). Under optimizedconditions (2 U ml⁻¹, thrombin; 0.2 mg-ml⁻¹ p120; 4 hours, 4° C., with 1mM EGTA and 1 mM MgCl₂) it was possible to generate preparations ofthrombin cleaved p120 which contained 15% uncut p120, 50% cut at theauthentic amino-terminal thrombin site and 35% with an additionalapproximately 40 residues cleaved. In these experiments thrombin actionwas terminated by the addition of 100 nM N-acetyl D-Phe Pro-2-amido-5guanidino butane boronic acid; a potent thrombin inhibitor (Sigma).Throughout this work, preparations of partially cleaved p120 were usedparallel with totally uncleaved p120. It was clear that: (A) all three120's bound to p101 with very similar affinity, (B) p101 bound to thep120 mixture was activated by Gβγs to a similar extent to p101 bound touncleaved p120 and, (C) both the uncleaved and partially cleaved p120swere virtually insensitive to Gβγ subunits in the absence of p101(although complete thrombin cleavage tended to reduce overall p120catalytic activity by 20-30% and increase the 1.7 fold apparentactivation by Gβγ in the absence of p101 to about 2.5 fold).

Porcine (EE)-p101 was purified from frozen pellets of Sf9 cells asfollows. Cells from 2 liters of infected Sf9 culture were sonicated into50 mls of 0.12M NaCl; 1MM MgCl_(2;) 25 mM HEPES, pH 7.4, 4° C.; 1 mMEGTA plus antiproteases I as described above. After centrifugation(120,000×g, 4° C., 40 minutes), the supernatant was removed (asdescribed above), supplemented with 1% w/v, Triton X100, 0.4% sodiumcholate, 0.4M NaCl (final concentrations), and mixed with 2 mls ofpacked protein G sepharose covalently cross-linked to an α-(myc)irrelevant, monoclonal antibody (washed in an equivalent solution).After 30 minutes mixing at 4° C., the beads were sedimented and thesupernatant was removed and mixed with 1 ml of protein G Sepharosecovalently cross-linked to an α-(EE) monoclonal antibody (equilibratedin an equivalent solution). After 2 hours mixing at 4° C. the beads werewashed as described below for U937 cell α-(EE) immunoprecipitations,except (a) the washes were in a 20 ml centrifuge tube, and (b) the beadswere finally washed 3× with buffer H, comprised of buffer G with 1%,w/v, Triton X-100. `p101 blank` (p101C) preparations used in someexperiments were prepared from either wild-type baculo-virus infected oruninfected cells exactly as described above.

p101/p120 heterodimers formed in vivo by co-infection of Sf9 cells withboth forms of recombinant baculo-virus were purified as described for(EE)-p101 except the immunoprecipitates were washed 4× with buffer I, 1,w/v, Triton X-100; 0.15M NaCl; 20 mM HEPES, pH 7.4, 4° C.; 1 mM EGTA; 2×with buffer J, comprised of buffer I supplemented with 0.4M NaCl (finalconcentration), then 3× with buffer G before being eluted with 1 bedvolume of 150 μg ml⁻¹ (final concentration) of (EE)-peptide in buffer G.The (EE)-peptide, amino-terminal-acetylated EYMPTD, has a very highaffinity for the α-(EE) monoclonal antibody. After the beads wereincubated with (EE)-peptide, on ice, for 40 minutes, the supernatant wasremoved. Aliquots of the eluted proteins were diluted and assayed forPI3K activity (see above) or mixed with SDS sample buffer directly.

In in vitro reconstitution experiments, p120 preparations in buffer Gsupplemented with 1k, w/v, Triton X-100 (now equivalent to buffer H)were mixed with (EE)-p101 (10:1 molar ratio of protein) still bound tothe protein G matrix, mixed for 2 hours (end on end at 4° C.), thenwashed and eluted with (EE)-peptide as described for the purification ofp101/p120-PI3K reconstituted in vivo, in the Example below.

B. RESULTS

Single-step purifications utilizing the tags yielded purified proteinpreparations as assessed by Coomassie staining; their apparent sizesmatched expectation. Further, both were correctly recognized in Westernblots by specific, COOH Terminal directed and internal-sequencedirected, antipeptide, rabbit polyclonal sera (not shown) indicatingtheir authenticity.

p120 bound tightly, and in 1:1 molar stoichiometry, to p101 both (a) invitro, when both proteins had been independently purified, mixed (withp101 still associated with the protein G matrix used to isolate it) thenwashed stringently before being eluted with (EE)-peptide or, (b) invivo, when Sf9 cells were co-infected with both forms of rbv andproteins were purified via the p101 tag (data not shown).

Free, purified p120 was a PtdIns(4,5)P₂ -selective, wortmannin-sensitivePI3K. Gβγ had a small and bi-modal effect on free p120 PI3K activity. Anequimolar mixture (final total concentration of 2 μM) of Gα's o, i₁, i₂and i₃ bound to GDP and in the presence of aluminum fluoride had nosignificant effect on free p120 PI3K activity (this was a preparation ofGαs which, when added in a 1.5 fold molar excess, could completelyinhibit the effects of Gβγs on PI3K). Tyrosine phosphorylated peptidesable to activate p85/p110-PI3K family members also had no detectableeffect on p120 PI3K activity. When bound to p101 (either in vivo or invitro) p120's, PI3K activity could be activated greater than 100× by Gβγsubunits. Tyrosine phosphorylated peptides and Gα-GDP/aluminum fluoridehad no effect on Gβγ activated, or basal, p101/p120 PI3K activity. Inthe absence of Gβγs the specific activity of p120 in a p101/p120 complexis lower than the specific activity of free p120 but is increasedgreater than 50× in their presence (see FIG. 7).

Comparison of the specific catalytic activities of pigneutrophil-derived PI3K (B) and the Sf9-derived p101/p120 heterodimers,under identical, Gβγ-stimulated, assay conditions, showed the Sf9derived material to have a 2× higher activity per mg protein. Thisresult is not inconsistent with the likely `age at assay` of a PI3Kpreparation derived from circulating neutrophils via a 4 daypurification protocol and indicates the bulk of the recombinant PI3K iscorrectly folded and that any critical post-translational modificationsmust be in place.

XII. EXAMPLE Expression of porcine p101 and p120 in mammalian cells

A family of mammalian expression vectors were constructed that enabledtransient expression of amino-terminal epitope-tagged forms (either(myc) or (EE)) of porcine p101 and p120. This example describes theproduction of purified recombinant p101 and p120 fusion proteins frommammalian cells, and subsequent analysis of their properties.

A. MATERIALS AND METHODS

1. CELL CULTURE

U937 cells were grown in RPMI 1640 with 10%, v/v, heat-inactivated(HI)-FBS and diluted 4 fold every 2 days. Cos-7 cells were grown in DMEM10% HI-FBS.

2. CONSTRUCTION OF EXPRESSION VECTORS

The use of N-terminal PCR and appropriate restriction sites allowed theporcine p120 and p101 ORFs to be manipulated into a form where theycould be inserted in frame into various expression vectors (in each casethe first amino acid encoded after the N-terminal tag was the startmethionine. The 3'-untranslated resin of the p120 cDNA was used in fulland that of the p101 cDNA truncated at a BamHI site (nucleotide 192,FIG. 1). The vectors used for cytomegalovirus-driven (CMV) expression inmammalian cells were (A) pCMV(EE) (encoding an N-terminal MEEEEFMPMPMEF(SEQ ID NO:7) or MEEEEFMPMEFSS (SEQ ID NO:8) `EE-tag` for p101 or p120expression, respectively) and (B) pCMV (myc) (encoding an N-terminalMEQKLISEEDLEF (SEQ ID NO:9)or MEQKLISEEDLEFSS (SEQ ID NO:10) `myc-tag`for p101 or p120 expression, respectively). All vectors wereN-terminally sequenced before use.

3. U937 TRANSFECTION PROTOCOLS

Exponentially growing U937 cells (diluted 12 hours previously) werewashed 2× with PBS and resuspended in sterile electroporation medium(EM) containing; 30 mM NaCl, 0.12M KCl, 8.1 mM Na₂ HPO₄, 1.46 mM KH₂ PO₄and 5 mM MgCl₂, at room temperature. Circular plasmid DNA was added in1× EM to the cells to produce a 0.5 ml final volume containing 1.4 ×10⁷cells and 40 μg total DNA (usually made up to 40 μg with an expressionplasmid with the same promoter and expressing a similarly tagged butirrelevant protein) and were transferred to a cuvette (0.4 cms gap,BioRad). After 15 minutes standing at room temperature the cells wereelectroporated (1 pulse at 280 V and 960 μF, with a BioRad Gene pulser;time constants were typically 18 msec) then placed on ice for a further8 minutes before being diluted into 5 mls of RPMI, 10% HI-FBS. Afterstanding for 5 minutes, to allow dead cells to clump together, the cellswere diluted with 35 ml of RPMI, 10% HI-FBS, supplemented withpenicillin and streptomycin, then TPA and ZnCl₂ were added (both ofwhich substantially amplify expression from CMV promoters in U937 cells)to final concentrations of 5×10⁸ M and 200 μM, respectively. If thecells were to be labelled with ³⁵ S!-methionine (trans-label, ICN) thenthe RPMI used after the electroporation was methionine-, andleucine-free and contained 2 mM NaHCO₃ and 25 mM HEPES and 10% dialyzedHI-FBS and the cells resuspended in a final volume of 10 mls with 20μCi/ml ³⁵ S!-methionine/leucine (phs TPA and ZnCl₂, as above) After 12hours (either with or without ³⁵ S!) the cell suspensions were mixedwith di-isopropyl fluorophosphate (1 mM final concentration), left for 5minutes, then collected by centrifugation, washed 1× with PBS, and lysedfor immunoprecipitation. (EE)-epitope tagged proteins wereimmunoprecipitated from U937 cell lysates as follows. Cells from 1electroporation were lysed into 1.25 mls of lysis buffer containing 1%,w/v, Triton X-100, 25 mM HEPES, pH 7.4, 4°C.; 1 mM EGTA; 1 mM MgCl₂ ;0.15 m NaCl and 0.1 mM PMSF, 10 μg ml⁻¹ leupeptin, 10 μgml⁻¹ aprotinin,10 μgml⁻¹ antipain 10 μgml⁻¹ pepstatin A and 10 μgml⁻¹ of bestatin(henceforth known as antiproteases I). The lysates were centrifuged(12,000 rpm, 0° C., 15 minutes). The resulting supernatants was removedand mixed with 4M NaCl (0.1 ml), 20% sodium cholate (25 μl) and proteinG sepharose (40 μl packed volume) covalently coupled to an irrelevant,isotype-matched, mouse monoclonal antibody (at approx. 5-10 mg-ml⁻¹sepharose). The supernatants were mixed end over end for 20 minutes,then the beads were sedimented and the supernatant transferred toanother tube with protein G sepharose beads (10 μl, packed) covalentlycrosslinked to α- (EE) -mouse monoclonal antibody (at approx. 5-10mg-ml⁻¹ sepharose). After 2 hours mixing at 4° C. the immunoprecipitateswere washed 5× with 1.0%, w/v, Triton X-100; 0.4% cholate; 0.004% SDS;0.4M NaCl; 1 mM EGTA; 25 mM HEPES, pH 7.4, at 0° C.; 1× with 0.5M LiCl,0.1M Tris, pH 8.0, 4° C.; 2× with 0.12M NaCl; 1 mM EGTA; 25 mM HEPES, pH7.4, at 0° C. The last buffer wash was supplemented with 1 mM DTT (finalwash buffer). If the immunoprecipitates were prepared from ³⁵S!-methionine labelled cells the last wash was omitted and the beadswere boiled in SDS sample buffer. Otherwise, the samples were assayedfor PI3K activity as described below.

4. COS-7 CELL TRANSFECTIONS

Exponentially growing Cos-7 cells were trypsinized/replated at about50-70% confluence 3 hours prior to transfection. At the time oftransfection they were again trypsinized, diluted into DMEM 10% FBS,counted, washed 2× in PBS and resuspended in EM (1×10⁷ per cuvette)mixed with circular plasmid DNA (40 μg total per cuvette, made up ofcombinations of 10 μg of EXV-(EE)-β₁, 10 μg of EXV-(myc)-τ₂, 10 μg ofpCMV-(myc)-p120 or 10-40 μg of an irrelevant mammalian expressionvector), to give a final volume of 500 μl and then transferred to anelectroporation cuvette (0.4 cms gap, BioRad). After 10 minutes at roomtemperature the cells were electroporated (250 V, 960 μF), placed on icefor 8 minutes then diluted into DMEM 10% FBS. Aggregated cells wereallowed to clump and were avoided as the cells were aliquoted into 6 cmdishes (four from each cuvette).

After 48 hours, the four dishes from each treatment were washed intoHEPES-buffered DMEM containing 1 mM NaHCO₃ and 0.2% fatty acid-free BSA.After 10 hours two replicate dishes were harvested for Western blotting(with α-(myc) monoclonal antibody as the 10 detection reagent,α-mouse-HRP as the 2° detection system and quantitation by ECL anddensiometric scanning). Two dishes were washed into phosphate-free,DMEM, with 1 mM NaHCO₃ and 0.2% fatty acid-free BSA, then incubated fora further 90 minutes at 37° C. with 300 μCi ³² P!-Pi per dish (in 4mls). Media was aspirated and 1 ml of ice-cold 1M HCl was added. Thecells were scraped, removed and the dishes washed with 1.33 mls ofmethanol. The HCl and methanol washes were pooled with 2.66 mls ofchloroform (to yield a `Folch` two phase, solvent distribution) mixedand centrifuged. The lower phases were removed and mixed with 1.95 mlsof fresh upper phase (see above) containing 0.5 mM EDTA and 1 mMtetrabutylammonium sulphate. After mixing and centrifugation, the lowerphases were removed, dried down, deacylated and prepared for analysis byanion-exchange HPLC (Stephens et al., 1991, Nature. Lond. 351:33-39, thedisclosure of which is incorporated herein by reference in itsentirety).

B. RESULTS

When transiently expressed in U937 cells, (EE)-p101 and (EE)-p120 couldbe specifically immunoprecipitated from ³⁵ S!-methionine labelled cellsin approximately equal amounts (allowing for their relative complimentof methionines; 8:25 respectively, data not shown). Stringently washedα-(EE)-p101 immunoprecipitates contained a wortmannin-sensitive,PtdIns(4,5)P₂ -selective, Gβγ-sensitive PI3K activity that was absent incontrols using either an irrelevant monoclonal antibody for theimmunoprecipitation, or a cDNA encoding an (EE)-tagged irrelevantprotein which was expressed, as judged by ³⁵ S!-methionine labelling, tosimilar levels as (EE)-p101.

The activation by Gβγτs could be blocked by preincubation with a 2 foldmolar excess of Gα-GDP. Further, the PI3K activity in these p101immunoprecipitates was insensitive to Gα-GDP/aluminum fluoride.Co-transfection of (myc)-p120 with (EE)-p101 did not increase the PI3Kactivity specifically recovered in α-(EE) immunoprecipitates. Indeed itdecreased, probably because the expression of (EE)-p101 was relativelylower in the presence of (myc)-p120 expression vectors. In contrast,cells transfected with (EE)-p120 α-(EE)-immunoprecipitates containedbarely detectable PI3K activity either in the presence or absence ofGβγs. These cells contained comparable moles of ³⁵ S!-methioninelabelled p120 as there was (EE)-p101 in α-(EE)-immunoprecipitates from(EE)-p101 transfected cells. Co-transfection with (myc)-p101 resulted ina substantial increase in, specifically, the Gβγτ-stimulated PI3Kactivity that could be recovered, despite a fall in the expression of(EE)-p120 (as judged by ³⁵ S!-methionine labelling) (see FIG. 8). Thisdata indicates that U937 cells (human) contain an endogenous PI3Kcatalytic subunit that can bind to a transiently expressed p101 (pig).When bound to p101 (pig) that endogenous catalytic subunit displayssubstantial regulation by Gβγs because all of the p120 present in theimmunoprecipitates is bound to p101.

In contrast, in α-(EE) immunoprecipitates from (EE)-p120 transfectedcells much of the p120 is unassociated with p101 and hence relativelyinactive (compared to that bound to p101 and in the presence of Gβγs).However, this PI3K activity, when assayed in the presence of Gβγs, issubstantially amplified by co-transfection with (myc)-p101. Thealternative explanation for these data--that the p120 is `denatured`(although soluble and capable of being immunoprecipitated) unlessexpressed in the presence of p101--is unlikely in view of the dataobtained with independently Sf9-purified, derived proteins.

To test whether the p101/p120 PI3K could be activated by Gβγs andproduce PtdIns(3,4,5)P₃ in vivo, we transiently expressed variouscombinations of (myc)-γ₂, (EE)-β₁, (myc)-p101 and (myc)-p120 in Cos-7cells and measured their effects on the levels of ³⁵P!-phosphoinositides in cells 48 hours after transfection (see FIG. 9).p120 only produced significant increases in PtdIns(3,4,5)P₃ andPtdIns(3,4)P₂ in a β₁ γ₂ -dependent fashion in the presence of p101.This pattern of results could not be explained by changes in therelative expression of the different cDNAs when introduced incombinations (see FIG. 9).

XIII. DEPOSIT OF CLONES

The following microorganisms or clones were deposited with the AmericanType Culture Collection (ATCC), Rockville, Maryland, on the datesindicated and were assigned the indicated accession number:

    ______________________________________    Clone        ATCC Access. No.                              Date of Deposit    ______________________________________    pCMV3mycp101 97636        12/27/96    pCMV3mycp120 97637        12/27/96    ______________________________________

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 14    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4692 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CCGTGCGCCCCTCAAGACTAATGGACCCCCGGCTCAGGAATCGCACGAGGCAGGCTCACA60    CCCGAGGCCCATGGAAGTTCCCAGGCCAGGGGTCAAGTTGGAACCGAAGCTGCTGCCAGC120    TTACACCACAGCCACAGCAACTTGGGATCTGAGCTGCATCTGTGACCTACACCACAGCTC180    ACGGCAATGCTGGATTCCCAACACACCAAGTGGGGCCAGGGATCGAACCCGCATCCTCTT240    GGACAGTAGTCAGATTCATTACCACTGAGCCATTGACAGGAACTCCAGGGGCAGGGGGGA300    GTCTCTTGTTTTTGGCTCCTCCCGACACCTGGTGAAATGGACCAGCGCAGGCACCCCTTT360    CCAGTGGCTGTCCCAGGCGATGACTCAGGATGCAGCCAGGGGCCACGACGTGCACGGAGG420    ACCGCATCCAGCACGCCCTGGAGCGCTGCTTGCACGGGCTCAGCCTCAGCCGCCGCTCCA480    CCTCCTGGTCAGCTGGGCTGTGTCTAAACTGTTGGAGCCTGCAGGAGCTGGTCAGCAGGG540    ATCCGGGCCACTTCCTTATCCTCCTGGAGCAGATCCTGCAGAAAACCCGAGAGGTCCAAG600    AGAAGGGCACCTATGACCTCCTCGCGCCCCTGGCCCTGCTCTTCTATTCTACTGTCCTCT660    GTACGCCACACTTCCCGCCAGACTCAGATCTCCTTCTGAAAGCAGCCAGAACCTACCACC720    GATTCCTGACCTGGCCGGTTCCGTACTGCAGCATCTGCCAGGAACTGCTCACCTTCATCG780    ATGCTGAGCTGAAGGCCCCAGGAATCTCCTACCAGCGACTGGTGAGGGCGGAGCAGGGCC840    TGTCCACAAGGAGTCACCGCAGCTCCACCGTCACGGTGCTCTTGGTGAACCCCGTGGAGG900    TGCAGGCTGAGTTCCTTGACGTGGCCGACAAGCTGAGCACACCAGGGCCCTCGCCGCACA960    GCGCCTACATCACCCTGCTCCTGCATGCCTTCCAGGCCACCTTTGGGGCCCACTGTGACC1020    TCTCTGGTCTGCACCGCAGGTTGCAGTCCAAGACCCTGGCAGAGCTCGAGGCCATCTTCA1080    CGGAGACAGCCGAGGCACAGGAGCTGGCCTCAGGCATCGGGGATGCAGCTGAGGCCCGGC1140    AGTGGCTCAGGACCAAGCTGCAGGCGGTGGGAGAGAAGGCCGGCTTCCCTGGTGTCTTAG1200    ACACCGCCAAACCTGGCAAGCTCCGCACCATCCCCATCCCGGTCGCCAGGTGCTACACCT1260    ACAGCTGGAACCAGGACAGCTTCGACATCCTGCAGGAAATCCTGCTCAAGGAGCAGGAGC1320    TGCTCCAGCCAGAGATCCTGGACGACGAGGAGGACGAGGACGAGGAGGACGAGGAAGAGG1380    ACTTGGACGCCGACGGCCACTGCGCGGAGAGGGACTCCGTGCTCTCCACCGGCTCGGCGG1440    CCTCCCACGCCTCCACGCTGTCCCTGGCCTCGTCCCAGGCCTCGGGGCCCACGCTCTCCC1500    GCCAGTTGCTGACCTCCTTCGTCTCGGGCCTCTCGGATGGCGTGGACAGCGGCTACATGG1560    AGGACATCGAGGAGAGCGCCTACGAGCGGCCCCGGAGGCCTGGCGGCCACGAGCGCCGGG1620    GCCACCGCCGGCCCGGGCAGAAGTTCAACAGGATCTATAAACTCTTCAAGAGCACCAGCC1680    AGATGGTGCTGCGGAGGGACTCGCGCAGCCTGGAGGGCAGCCCGGACAGCGGCCCGCCCC1740    TGCGTCGGGCCGGCAGCCTCTGCAGCCCCCTGGACAGCCCGACCCTGCCCCCGTCCCGGG1800    CCCAGGGCTCCCGCTCGCTGCCCCAGCCCAAGCTCAGCCCCCAGCTGCCCGGCTGGCTCC1860    TGGCCCCCGCCTCCCGCCACCAGCGCCGCCGCCCCTTCCTGAGCGGGGACGAGGACCCCA1920    AGGCTTCCACGCTGCGTGTCGTGGTCTTCGGCTCGGATCGGATCTCGGGGAAGGTGGTCC1980    GGGCTTACAGCAACCTGCGGCGGCTGGAGAACAACCGTCCTCTCCTCACACGGTTCTTCA2040    AGCTACAGTTCTTCTACGTGCCTGTCAAGCGGAGCCGTGGGACAGGCACCCCCACCAGCC2100    CAGCCCCTCGGAGCCAGACGCCCCCCCTCCCCACAGACGCCCCGAGGCACCCGGGCCCTG2160    CAGAGCTGGGCGCCGCCCCCTGGGAGGAGAGCACCAATGACATCTCCCACTACCTCGGCA2220    TGCTCGACCCCTGGTACGAGCGAAACGTCCTGGGCCTCATGCACCTGCCTCCTGAAGTCC2280    TGTGCCAGTCCCTGAAGGCTGAGCCCCGGCCCCTGGAGGGCTCCCCTGCCCAGCTGCCCA2340    TCCTGGCGGACATGCTGCTCTACTACTGCCGCTTCGCTGCCCGGCCGGTGCTGCTGCAGG2400    TCTATCAGACCGAGCTGACCTTCATCACCGGGGAGAAGACGACGGAGATCTTCATCCACT2460    CCCTGGAGCTGGGCCACTCTGCTGCCACACGTGCCATCAAGGCTTCGGGTCCTGGCAGCA2520    AGCGGCTGGGCATCGATGGTGACCGGGAGGCCGTCCCTCTAACACTACAGATAATTTACA2580    GCAAGGGGGCCATCAGCGGCCGGAGTCGCTGGAGCAACATGGAAAAGCTCTGCACCTCTG2640    TCAACCTCAGCAAGGCCTGCCGGCAGCAGGAGGAGCTAGACTCCAGCACAGAGGCCCTGA2700    CGCTAAACCTGACAGAAGTGGTGAAAAGACAGACCCCTAAATCCAAGAAGGGCTTTAACC2760    AGATCAGCACCTCGCAGATCAAAGTGGACAAGGTGCAGATCATCGGCTCTAACAGCTGCC2820    CCTTTGCCGTGTGTCTGGACCAGGACGAGAGGAAGATCCTGCAGAGTGTCATCAGGTGCG2880    AGGTCTCGCCCTGCTACAAGCCTGAGAAGAGCAGCCTCTGCCCCCCACCCCAGAGGCCCT2940    CCTACCCGCCAGCGCCGGCCACGCCCGACCTCTGCTCCCTGCTCTGCCTGCCCATCATGA3000    CTTTCAGCGGAGCTCTGCCCTAGCCGCCACCCTGCACCAGCCTGGACAGGGAGCCGGGGG3060    GCAGCCTCCTCGGAGCCCCCTCCCCAGAAGACTGGCGGCTGAGAGGGTCGTGCTCCCTGT3120    GGAGAACAGAGGGGCCGTGTACTGGGTCAGGGTCCCGCTGTGGGCCCTGCAGCAGCAAGA3180    GCGGGGGCTGCTGGGGCCTCAGGGCTCTGTTTGGGCGAGAAGCAGGCATTAGGGAGAGGG3240    GCCTGGCCCCACGGCTCTCAGCTTCCTCACGGTAGCGGAGAGAGGGATGGGTGAGCTTGA3300    CCTCAAGGCCCTGGCCTCCAGTGGGGGTCCAGGATCCTTTCTGGAAGGAAGATCCCAAGG3360    CGCTGGTGCTCTGGGGTGTGGTGTTAGGGGCTCCCCCCCCAGCCCTGGGCCAGGGCCCCC3420    CCGTTACTTTGTCAGAGACTTGGGGATCCTGTGTCTGGAGGGTCAAGTCCCCCTCCCTGG3480    GGGTTCAAGCAGTGGAAGTATGGTTGCGACTTTTCTGACGTTGGTGCAATCCCCGCCCCC3540    ACCTCAACCCCCCCACAAAAAAACCCCTTCTCTCTTTCAAGTTCCCTGGGTCTTCTGTGA3600    AACAGCACTAACACTTGACCTGGCTGTGCCAGCACTTGGAACAGATGCTCCCTGGATCGA3660    GAGCCTTGGGAGACAGGACAAGCTTAGGTTCGGTGGTGGCTCAGTTACCTTCTAGCGAAA3720    TGAGCAGAAGGAGGTGAATTGGCTCCTTCGAGGCTCCCCTACCTGGGCACTAAGATGGGG3780    GGAATAAGGCCGCCTTAAAGGGTTGGGGTGATGTCGTCTGCAAAGCGCCTGGCCCAGTGG3840    CCGGCTGGTAGCAAGGTGCGGCCTCACCCTCTGGGCGTCGACTCCCTCGTGTGGCGGGAG3900    GCTAAAAGGATGCCCTGCCCCCGTGATGCTGTCATTCCCTCCTTCCAATTCACTGATGAG3960    GCAGGACCCAGACTGAGGGGGTGAGGGGCGCACAGTTCTACCTTTGAAGGAGGAAGTGCC4020    TTGATCAGAGTAAGAGGAGGGTGGCCCAGGCGCCCCCAACCGCCCCCTCCTCCTCTCCCA4080    GGTTGGCCCCTGTGCCTCCCACTCCCATCTCACTCCTTGGGCTGGCGCACATCACGGGCA4140    CAGTCCTCCAGCCCCACAGTTCACTGGTACCATGGCCCCTGGGTCGGTTCGCAGAGGATG4200    GAGGATAAGACTTGCCTCGAGAACTTGGGTCTGATGGGGAAACCGGGTGATGGAAATGAT4260    TCCGGAAGATTAAAACCTCCCAGGTTCAAGTGTCGGAGAACCGCCCCCACAACCGGACTA4320    GGTTGGTAGGGAGAGGGCAGGGCTTGGGCCCGGGATTTGGACTAGGAGAGGCGGGGGGAG4380    GTAACCAGAGAAGCAAGACAGTTGTATCCCCGCAAAAGACCCTTCCCCGCCCCTCCCCTC4440    CTGCTCTGGCTCCATCTGCTTCAAAGGGTCTGGGCTTTAGGAGCCCGTGGTGCCCAGCGC4500    AGCGTACTCAGGACTCGAGAGACGCGGACCGTGCCAGTTCCCACCCTGTGCCACTCCAGG4560    CCCCAGGGAGGGGTTTGCAATATACCCTCAACGTTTTTGTGTGTGTGGTAAGGTCGTCCT4620    AGGACCCCAAATGGAATTTAACGTTATTGTCAAATAAAACTTGATTTGTCTTGGAAAAAA4680    AAAAAAAAAAAA4692    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 877 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetGlnProGlyAlaThrThrCysThrGluAspArgIleGlnHisAla    151015    LeuGluArgCysLeuHisGlyLeuSerLeuSerArgArgSerThrSer    202530    TrpSerAlaGlyLeuCysLeuAsnCysTrpSerLeuGlnGluLeuVal    354045    SerArgAspProGlyHisPheLeuIleLeuLeuGluGlnIleLeuGln    505560    LysThrArgGluValGlnGluLysGlyThrTyrAspLeuLeuAlaPro    65707580    LeuAlaLeuLeuPheTyrSerThrValLeuCysThrProHisPhePro    859095    ProAspSerAspLeuLeuLeuLysAlaAlaArgThrTyrHisArgPhe    100105110    LeuThrTrpProValProTyrCysSerIleCysGlnGluLeuLeuThr    115120125    PheIleAspAlaGluLeuLysAlaProGlyIleSerTyrGlnArgLeu    130135140    ValArgAlaGluGlnGlyLeuSerThrArgSerHisArgSerSerThr    145150155160    ValThrValLeuLeuValAsnProValGluValGlnAlaGluPheLeu    165170175    AspValAlaAspLysLeuSerThrProGlyProSerProHisSerAla    180185190    TyrIleThrLeuLeuLeuHisAlaPheGlnAlaThrPheGlyAlaHis    195200205    CysAspLeuSerGlyLeuHisArgArgLeuGlnSerLysThrLeuAla    210215220    GluLeuGluAlaIlePheThrGluThrAlaGluAlaGlnGluLeuAla    225230235240    SerGlyIleGlyAspAlaAlaGluAlaArgGlnTrpLeuArgThrLys    245250255    LeuGlnAlaValGlyGluLysAlaGlyPheProGlyValLeuAspThr    260265270    AlaLysProGlyLysLeuArgThrIleProIleProValAlaArgCys    275280285    TyrThrTyrSerTrpAsnGlnAspSerPheAspIleLeuGlnGluIle    290295300    LeuLeuLysGluGlnGluLeuLeuGlnProGluIleLeuAspAspGlu    305310315320    GluAspGluAspGluGluAspGluGluGluAspLeuAspAlaAspGly    325330335    HisCysAlaGluArgAspSerValLeuSerThrGlySerAlaAlaSer    340345350    HisAlaSerThrLeuSerLeuAlaSerSerGlnAlaSerGlyProThr    355360365    LeuSerArgGlnLeuLeuThrSerPheValSerGlyLeuSerAspGly    370375380    ValAspSerGlyTyrMetGluAspIleGluGluSerAlaTyrGluArg    385390395400    ProArgArgProGlyGlyHisGluArgArgGlyHisArgArgProGly    405410415    GlnLysPheAsnArgIleTyrLysLeuPheLysSerThrSerGlnMet    420425430    ValLeuArgArgAspSerArgSerLeuGluGlySerProAspSerGly    435440445    ProProLeuArgArgAlaGlySerLeuCysSerProLeuAspSerPro    450455460    ThrLeuProProSerArgAlaGlnGlySerArgSerLeuProGlnPro    465470475480    LysLeuSerProGlnLeuProGlyTrpLeuLeuAlaProAlaSerArg    485490495    HisGlnArgArgArgProPheLeuSerGlyAspGluAspProLysAla    500505510    SerThrLeuArgValValValPheGlySerAspArgIleSerGlyLys    515520525    ValValArgAlaTyrSerAsnLeuArgArgLeuGluAsnAsnArgPro    530535540    LeuLeuThrArgPhePheLysLeuGlnPhePheTyrValProValLys    545550555560    ArgSerArgGlyThrGlyThrProThrSerProAlaProArgSerGln    565570575    ThrProProLeuProThrAspAlaProArgHisProGlyProAlaGlu    580585590    LeuGlyAlaAlaProTrpGluGluSerThrAsnAspIleSerHisTyr    595600605    LeuGlyMetLeuAspProTrpTyrGluArgAsnValLeuGlyLeuMet    610615620    HisLeuProProGluValLeuCysGlnSerLeuLysAlaGluProArg    625630635640    ProLeuGluGlySerProAlaGlnLeuProIleLeuAlaAspMetLeu    645650655    LeuTyrTyrCysArgPheAlaAlaArgProValLeuLeuGlnValTyr    660665670    GlnThrGluLeuThrPheIleThrGlyGluLysThrThrGluIlePhe    675680685    IleHisSerLeuGluLeuGlyHisSerAlaAlaThrArgAlaIleLys    690695700    AlaSerGlyProGlySerLysArgLeuGlyIleAspGlyAspArgGlu    705710715720    AlaValProLeuThrLeuGlnIleIleTyrSerLysGlyAlaIleSer    725730735    GlyArgSerArgTrpSerAsnMetGluLysLeuCysThrSerValAsn    740745750    LeuSerLysAlaCysArgGlnGlnGluGluLeuAspSerSerThrGlu    755760765    AlaLeuThrLeuAsnLeuThrGluValValLysArgGlnThrProLys    770775780    SerLysLysGlyPheAsnGlnIleSerThrSerGlnIleLysValAsp    785790795800    LysValGlnIleIleGlySerAsnSerCysProPheAlaValCysLeu    805810815    AspGlnAspGluArgLysIleLeuGlnSerValIleArgCysGluVal    820825830    SerProCysTyrLysProGluLysSerSerLeuCysProProProGln    835840845    ArgProSerTyrProProAlaProAlaThrProAspLeuCysSerLeu    850855860    LeuCysLeuProIleMetThrPheSerGlyAlaLeuPro    865870875    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3808 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGCACGAGGAATTTGTTTTGTTTTCAGAAATTAAACAAATGATCCTTCAGCATCATCACC60    TCCGCTGCTTTATCAGGTCGCATAGGGCATGGAGCTGGAGAACTATGAACAGCCCGTGGT120    GCTGAGAGAGGACAACCGCCGCAGGCGTCGGAGGATGAAGCCGCGCAGCACGGCAGCCAG180    CCTGTCCTCCATGGAGCTCATCCCCATCGAGTTTGTTTTGGCCACCAGCCAGCGCAACAC240    CAAGACCCCCGAAACGGCACTGCTGCACGTGGCCGGCCACGGCAATGTGGAGAAGATGAA300    GGCCCAGGTGTTGTTGCGCGCGCTGGAGACGAGCGTTTCTTGGGACTTCTACCACCGGTT360    CGGCCCCGACCACTTCCTCCTGGTCTTCCAGAAGAAGGGGGAGTGGTACGAGATCTATGA420    CAAGTACCAGGTGGTGCAGACCCTGGACTGCCTGCGCTACTGGGAGGTGTTGCACCGCAG480    CCCCGGGCAGATCCACGTGGTCCAGCGGCACGCGCCCTCGGAGGAGACATTGGCCTTCCA540    GCGCCAGCTCAACGCCCTCATCGGCTACGACGTCACCGACGTCAGCAACGTGCATGACGA600    TGAGCTGGAGTTCACGCGGCGCCGCCTGGTCACCCCGCGCATGGCCGAGGTGGCCGGCCG660    CGACCCCAAGCTTTACGCCATGCACCCCTGGGTGACATCCAAGCCCCTCCCTGAGTACCT720    TCTGAAGAAGATCACTAACAACTGCGTCTTCATCGTCATTCACCGCAGCACCACCAGCCA780    GACCATCAAGGTCTCGGCCGATGACACCCCAGGCACCATCCTCCAGAGCTTCTTTACCAA840    GATGGCCAAGAAGAAATCTCTGATGGATATCCCTGAAAGCCAGAACGAACGGGACTTTGT900    GCTGCGCGTCTGCGGCCGGGATGAGTACCTGGTGGGTGAGACGCCCATCAAAAATTTCCA960    GTGGGTGAGGCAGTGCCTCAAGAATGGGGAGGAGATTCACCTTGTGCTGGACACTCCTCC1020    AGACCCAGCCCTGGACGAGGTGAGGAAGGAAGAGTGGCCGCTGGTGGATGACTGCACGGG1080    AGTCACTGGCTACCACGAGCAGCTGACCATCCACGGCAAGGACCATGAAAGTGTGTTCAC1140    CGTGTCCCTGTGGGACTGTGACCGCAAGTTCAGGGTCAAAATCAGAGGCATTGATATCCC1200    TGTCCTGCCCCGGACCGCTGACCTCACGGTGTTTGTGGAGGCAAACATCCAGTATGGGCA1260    GCAAGTCCTTTGCCAAAGGAGAACCAGCCCCAAACCCTTCACGGAGGAGGTGCTCTGGAA1320    CGTGTGGCTTGAGTTCAGTATTAAAATCAAAGACTTACCCAAAGGGGCTCTGCTGAACCT1380    CCAGATCTACTGCGGCAAAGCTCCAGCACTGTCTGGCAAGACCTCTGCAGAGATGCCCAG1440    TCCCGAGTCCAAAGGCAAAGCTCAGCTTCTGTACTATGTCAACCTATTGCTGATAGACCA1500    CCGCTTCCTCCTGCGCCATGGCGAGTATGTGCTCCACATGTGGCAGTTATCCGGGAAGGG1560    GGAAGACCAAGGGAGCTTCAATGCCGACAAGCTCACGTCGGGAACCAACCCGGACAAGGA1620    GGACTCAATGTCCATCTCCATTCTTCTGGACAATTACTGCCACCCCATAGCCTTGCCTAA1680    GCATCGGCCTACCCCTGACCCAGAAGGGGACCGGGTTCGGGCAGAAATGCCCAATCAGCT1740    TCGGAAGCAACTGGAGGCAATCATAGCCACGGATCCGCTTAACCCACTCACAGCTGAAGA1800    CAAAGAACTGCTCTGGCATTTCAGATATGAAAGCCTGAAGGATCCCAAAGCGTATCCTAA1860    GCTCTTTAGCTCGGTGAAATGGGGACAGCAAGAAATTGTGGCCAAAACATACCAATTATT1920    AGCCAAAAGGGAGGTCTGGGATCAGAGTGCTTTGGATGTGGGGTTAACCATGCAGCTCCT1980    GGACTGCAACTTCTCGGATGAAAACGTGAGAGCCATTGCAGTCCAGAAACTGGAGAGCTT2040    GGAGGATGATGACGTGCTCCATTACCTGCTCCAGCTGGTCCAGGCTGTGAAATTTGAACC2100    ATACCATGACAGTGCCCTAGCCAGATTTCTGCTGAAGCGTGGTTTAAGAAACAAGAGAAT2160    TGGTCACTTCTTGTTTTGGTTCTTGAGAAGTGAGATTGCCCAGTCTAGGCACTATCAGCA2220    GAGGTTTGCAGTGATCCTGGAAGCCTACCTGAGGGGCTGTGGCACAGCCATGCTGCACGA2280    CTTCACCCAGCAAGTCCAAGTAATTGACATGTTACAAAAAGTCACCATTGACATTAAATC2340    GCTCTCTGCTGAAAAGTATGACGTCAGTTCCCAAGTTATTTCCCAACTTAAGCAAAAGCT2400    TGAAAACCTACAGAATTTGAATCTCCCCCAAAGCTTTAGAGTTCCCTATGATCCTGGACT2460    GAAAGCCGGGGCACTGGTGATCGAAAAATGTAAAGTGATGGCCTCCAAGAAGAAGCCCCT2520    GTGGCTTGAGTTTAAATGTGCCGATCCTACGGCTCTATCAAATGAAACAATTGGAATTAT2580    CTTTAAACACGGTGACGATCTGCGCCAAGACATGCTTATTTTACAGATTCTACGAATCAT2640    GGAGTCCATTTGGGAGACCGAATCTTTGGATCTGTGCCTCCTGCCATATGGCTGCATTTC2700    AACTGGTGACAAAATAGGAATGATCGAGATCGTGAAGGACGCCACGACAATCGCCAAAAT2760    TCAGCAAAGCACAGTGGGCAACACGGGTGCCTTTAAAGATGAAGTCCTGAGTCACTGGCT2820    CAAAGAAAAATGCCCTATTGAAGAAAAGTTTCAGGCAGCTGTGGAGAGATTTGTTTATTC2880    CTGTGCCGGCTACTGTGTGGCAACCTTTGTTCTCGGAATAGGCGACAGACACAATGACAA2940    TATTATGATCTCAGAAACAGGAAATCTATTTCATATTGATTTCGGACACATTCTTGGGAA3000    TTACAAAAGTTTCCTGGGCATTAATAAAGAGAGGGTGCCATTTGTGCTAACCCCAGACTT3060    CCTGTTTGTGATGGGGACTTCTGGAAAGAAGACAAGTCTACACTTCCAGAAATTTCAGGA3120    TGTCTGCGTCAAGGCTTACCTAGCCCTTCGTCATCACACAAACCTACTGATCATCCTCTT3180    CTCCATGATGCTGATGACAGGAATGCCCCAGTTAACCAGCAAAGAAGACATTGAATACAT3240    TCGGGATGCCCTCACAGTGGGCAAAAGTGAGGAGGATGCTAAAAAGTATTTTCTGGATCA3300    GATTGAAGTTTGCAGAGACAAAGGATGGACCGTGCAGTTTAACTGGTTCTTACATCTTGT3360    TCTTGGCATCAAACAAGGGGAGAAGCATCCCGCATAAAACTTTGGGCCAAGAGTTAAAAC3420    CCAAGTTATTGTCCTAATGCTTTACGTCAGCAGGACAATCACCGAACTTGATGTCATGTA3480    GTGGGACATTATGAAAGCTGGCACTTGAGAAATATAGCTCTTCCCCTAACTGAACTCTTC3540    ACTGGAGAAAAACCTTGGCATGTTTAAGTAATGTTCAGTGTTAGGCTTATTTGCATGTTT3600    GTTTTTTCTCATGTGCCCCCTCAGTCATGTTGGAGACTGTTCTAAATTTAAGTGGCCTAA3660    TGACCTCTGAAGTTTCAACTTTCTTGGTACTGAGTGCTTCTGAAATTCTTTACAATAATT3720    GGTAACATCTATTGTCAGCTGGGTATCCTCTCAATTTTGGTTATCCTTGGGTTTCTCAAA3780    CTCCTTACAGGAAAAAAAAAAAAAAAAA3808    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1102 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetGluLeuGluAsnTyrGluGlnProValValLeuArgGluAspAsn    151015    ArgArgArgArgArgArgMetLysProArgSerThrAlaAlaSerLeu    202530    SerSerMetGluLeuIleProIleGluPheValLeuAlaThrSerGln    354045    ArgAsnThrLysThrProGluThrAlaLeuLeuHisValAlaGlyHis    505560    GlyAsnValGluLysMetLysAlaGlnValLeuLeuArgAlaLeuGlu    65707580    ThrSerValSerTrpAspPheTyrHisArgPheGlyProAspHisPhe    859095    LeuLeuValPheGlnLysLysGlyGluTrpTyrGluIleTyrAspLys    100105110    TyrGlnValValGlnThrLeuAspCysLeuArgTyrTrpGluValLeu    115120125    HisArgSerProGlyGlnIleHisValValGlnArgHisAlaProSer    130135140    GluGluThrLeuAlaPheGlnArgGlnLeuAsnAlaLeuIleGlyTyr    145150155160    AspValThrAspValSerAsnValHisAspAspGluLeuGluPheThr    165170175    ArgArgArgLeuValThrProArgMetAlaGluValAlaGlyArgAsp    180185190    ProLysLeuTyrAlaMetHisProTrpValThrSerLysProLeuPro    195200205    GluTyrLeuLeuLysLysIleThrAsnAsnCysValPheIleValIle    210215220    HisArgSerThrThrSerGlnThrIleLysValSerAlaAspAspThr    225230235240    ProGlyThrIleLeuGlnSerPhePheThrLysMetAlaLysLysLys    245250255    SerLeuMetAspIleProGluSerGlnAsnGluArgAspPheValLeu    260265270    ArgValCysGlyArgAspGluTyrLeuValGlyGluThrProIleLys    275280285    AsnPheGlnTrpValArgGlnCysLeuLysAsnGlyGluGluIleHis    290295300    LeuValLeuAspThrProProAspProAlaLeuAspGluValArgLys    305310315320    GluGluTrpProLeuValAspAspCysThrGlyValThrGlyTyrHis    325330335    GluGlnLeuThrIleHisGlyLysAspHisGluSerValPheThrVal    340345350    SerLeuTrpAspCysAspArgLysPheArgValLysIleArgGlyIle    355360365    AspIleProValLeuProArgThrAlaAspLeuThrValPheValGlu    370375380    AlaAsnIleGlnTyrGlyGlnGlnValLeuCysGlnArgArgThrSer    385390395400    ProLysProPheThrGluGluValLeuTrpAsnValTrpLeuGluPhe    405410415    SerIleLysIleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGln    420425430    IleTyrCysGlyLysAlaProAlaLeuSerGlyLysThrSerAlaGlu    435440445    MetProSerProGluSerLysGlyLysAlaGlnLeuLeuTyrTyrVal    450455460    AsnLeuLeuLeuIleAspHisArgPheLeuLeuArgHisGlyGluTyr    465470475480    ValLeuHisMetTrpGlnLeuSerGlyLysGlyGluAspGlnGlySer    485490495    PheAsnAlaAspLysLeuThrSerGlyThrAsnProAspLysGluAsp    500505510    SerMetSerIleSerIleLeuLeuAspAsnTyrCysHisProIleAla    515520525    LeuProLysHisArgProThrProAspProGluGlyAspArgValArg    530535540    AlaGluMetProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAla    545550555560    ThrAspProLeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrp    565570575    HisPheArgTyrGluSerLeuLysAspProLysAlaTyrProLysLeu    580585590    PheSerSerValLysTrpGlyGlnGlnGluIleValAlaLysThrTyr    595600605    GlnLeuLeuAlaLysArgGluValTrpAspGlnSerAlaLeuAspVal    610615620    GlyLeuThrMetGlnLeuLeuAspCysAsnPheSerAspGluAsnVal    625630635640    ArgAlaIleAlaValGlnLysLeuGluSerLeuGluAspAspAspVal    645650655    LeuHisTyrLeuLeuGlnLeuValGlnAlaValLysPheGluProTyr    660665670    HisAspSerAlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsn    675680685    LysArgIleGlyHisPheLeuPheTrpPheLeuArgSerGluIleAla    690695700    GlnSerArgHisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyr    705710715720    LeuArgGlyCysGlyThrAlaMetLeuHisAspPheThrGlnGlnVal    725730735    GlnValIleAspMetLeuGlnLysValThrIleAspIleLysSerLeu    740745750    SerAlaGluLysTyrAspValSerSerGlnValIleSerGlnLeuLys    755760765    GlnLysLeuGluAsnLeuGlnAsnLeuAsnLeuProGlnSerPheArg    770775780    ValProTyrAspProGlyLeuLysAlaGlyAlaLeuValIleGluLys    785790795800    CysLysValMetAlaSerLysLysLysProLeuTrpLeuGluPheLys    805810815    CysAlaAspProThrAlaLeuSerAsnGluThrIleGlyIleIlePhe    820825830    LysHisGlyAspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeu    835840845    ArgIleMetGluSerIleTrpGluThrGluSerLeuAspLeuCysLeu    850855860    LeuProTyrGlyCysIleSerThrGlyAspLysIleGlyMetIleGlu    865870875880    IleValLysAspAlaThrThrIleAlaLysIleGlnGlnSerThrVal    885890895    GlyAsnThrGlyAlaPheLysAspGluValLeuSerHisTrpLeuLys    900905910    GluLysCysProIleGluGluLysPheGlnAlaAlaValGluArgPhe    915920925    ValTyrSerCysAlaGlyTyrCysValAlaThrPheValLeuGlyIle    930935940    GlyAspArgHisAsnAspAsnIleMetIleSerGluThrGlyAsnLeu    945950955960    PheHisIleAspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeu    965970975    GlyIleAsnLysGluArgValProPheValLeuThrProAspPheLeu    980985990    PheValMetGlyThrSerGlyLysLysThrSerLeuHisPheGlnLys    99510001005    PheGlnAspValCysValLysAlaTyrLeuAlaLeuArgHisHisThr    101010151020    AsnLeuLeuIleIleLeuPheSerMetMetLeuMetThrGlyMetPro    1025103010351040    GlnLeuThrSerLysGluAspIleGluTyrIleArgAspAlaLeuThr    104510501055    ValGlyLysSerGluGluAspAlaLysLysTyrPheLeuAspGlnIle    106010651070    GluValCysArgAspLysGlyTrpThrValGlnPheAsnTrpPheLeu    107510801085    HisLeuValLeuGlyIleLysGlnGlyGluLysHisProAla    109010951100    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 36 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ix) FEATURE:    (A) NAME/KEY: Other    (B) LOCATION: 12    (D) OTHER INFORMATION: N = Inosine    (A) NAME/KEY: Other    (B) LOCATION: 21    (D) OTHER INFORMATION: N = Inosine    (A) NAME/KEY: Other    (B) LOCATION: 27    (D) OTHER INFORMATION: N = Inosine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CAYGAYTTYACNCARCARGTNCARGTNATHGAYATG36    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ix) FEATURE:    (A) NAME/KEY: Other    (B) LOCATION: 3    (D) OTHER INFORMATION: N = Inosine    (A) NAME/KEY: Other    (B) LOCATION: 18    (D) OTHER INFORMATION: N = Inosine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GCNTAYATGGARGAYATNGARGA23    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    MetGluGluGluGluPheMetProMetProMetGluPhe    1510    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetGluGluGluGluPheMetProMetGluPheSerSer    1510    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    MetGluGlnLysLeuIleSerGluGluAspLeuGluPhe    1510    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetGluGlnLysLeuIleSerGluGluAspLeuGluPheSerSer    151015    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3255 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AAGCTTACTCAGCGCATCTGCTACAGCATTTTATCTTCCCAAGAGCCCCATGAGGCGATG60    ACCCAGGATGCAGCCAGGGGCCACGACATGCACGGAGGACCGCATCCAGCATGCCCTGGA120    ACGCTGCCTGCATGGACTCAGCCTCAGCCGCCGCTCCACCTCCTGGTCAGCTGGGCTGTG180    TCTGAACTGCTGGAGCCTGCAGGAGCTGGTCAGCAGGGACCCGGGCCACTTCCTTATCCT240    CCTTGAGCAGATCCTGCAGAAGACCCGAGAGGTCCAGGAGAAGGGCACCTACGACCTGCT300    CACCCCGCTGGCCCTGCTCTTCTATTCCACTGTTCTTTGTACACCACACTTCCCACCAGA360    CTCGGATCTCCTTCTGAAGGCAGCCAGCACCTACCACCGGTTCCTGACCTGGCCTGTTCC420    TTACTGCAGCATCTGCCAGGAGCTGCTCACCTTCATTGATGCTGAACTCAAGGCCCCAGG480    GATCTCCTACCAGAGACTGGTGAGGGCTGAGCAGGGCCTGCCCATCAGGAGTCACCGCAG540    CTCCACCGTCACCGTGCTGCTGCTGAACCCAGTGGAAGTGCAGGCCGAGTTCCTTGCTGT600    AGCCAATAAGCTGAGTACGCCCGGACACTCGCCTCACAGTGCCTACACCACCCTGCTCCT660    GCACGCCTTCCAGGCCACCTTTGGGGCCCACTGTGACGTCCCGGGCCTGCACTGCAGGCT720    ACAGGCCAAGACCCTGGCAGAGCTTGAGGACATCTTCACGGAGACCGCAGAGGCACAGGA780    GCTGGCATCTGGCATCGGGGATGCTGCAGAGGCCCGGCGGTGGCTCAGGACCAAGCTGCA840    GGCGGTGGGAGAAAAAGCTGGCTTCCCTGGGGTGTTAGACACTGCAAAACCAGGGAAGCT900    CCACACCATCCCCATCCCTGTCGCCAGGTGCTACACCTACAGCTGGAGCCAGGACAGCTT960    TGACATCCTGCAGGAAATCCTGCTCAAGGAACAGGAGCTACTCCAGCCAGGGATCCTGGG1020    AGATGATGAAGAGGAGGAAGAGGAGGAGGAGGAGGTGGAGGAGGACTTGGAAACTGACGG1080    GCACTGTGCCGAGAGAGATTCCCTGCTCTCCACCAGCTCTTTGGCGTCCCATGACTCCAC1140    CTTGTCCCTTGCATCCTCCCAGGCCTCGGGGCCGGCCCTCTCGCGCCATCTGCTGACTTC1200    CTTTGTCTCAGGCCTCTCTGATGGCATGGACAGCGGCTACGTGGAGGACAGCGAGGAGAG1260    CTCCTCCGAGTGGCCTTGGAGGCGTGGCAGCCAGGAACGCCGAGGCCACCGCAGGCCTGG1320    GCAGAAGTTCATCAGGATCTATAAACTCTTCAAGAGCACCAGCCAGCTGGTACTGCGGAG1380    GGACTCTCGGAGCCTGGAGGGCAGCTCGGACACGGCCCTGCCCCTGAGGCGGGCAGGGAG1440    CCTCTGCAGCCCCCTGGACGAACCAGTATCACCCCCTTCCCGGGCCCAGCGCTCCCGCTC1500    CCTGCCCCAGCCCAAACTCGGTACCCAGCTGCCCAGCTGGCTTCTGGCCCCTGCTTCACG1560    CCCCCAGCGCCGCCGCCCCTTCCTGAGTGGAGATGAGGATCCCAAGGCTTCCACGCTACG1620    TGTTGTGGTCTTTGGCTCCGATCGGATTTCAGGGAAGGTGGCTCGGGCGTACAGCAACCT1680    TCGGCGGCTGGAGAACAATCGCCCACTCCTCACACGGTTCTTCAAACTTCAGTTCTTCTA1740    CGTGCCTGTGAAGCGAAGTCATGGGACCAGCCCTGGTGCCTGTCCACCCCCTCGGAGCCA1800    GACGCCCTCACCCCCGACAGACTCCCCTAGGCACGCCAGCCCTGGAGAGCTGGGCACCAC1860    CCCATGGGAGGAGAGCACCAATGACATCTCCCACTACCTCGGCATGCTGGACCCCTGGTA1920    TGAGCGCAATGTACTGGGCCTCATGCACCTGCCCCCTGAAGTCCTGTGCCAGCAGTCCCT1980    GAAGGCTGAAGCCCAGGCCCTGGAGGGCTCCCCAACCCAGCTGCCCATCCTGGCTGACAT2040    GCTACTCTACTACTGCCGCTTTGCCGCCAGACCGGTGCTGCTGCAACTCTATCAGACCGA2100    GCTGACCTTCATCACTGGGGAGAAGACGACAGAGATCTTCATCCACTCCTTGGAGCTGGG2160    TCACTCCGCTGCCACACGTGCCATCAAGGCGTCAGGTCCTGGCAGCAAGCGGCTGGGCAT2220    CGATGGCGACCGGGAGGCTGTTCCTCTAACACTACAGATTATTTACAGCAAGGGGGCCAT2280    CAGTGGACGAAGTCGCTGGAGCAACCTGGAGAAGGTCTGTACCTCCGTGAACCTCAACAA2340    GGCCTGCCGGAAGCAGGAGGAGCTGGATTCCAGCATGGAGGCCCTGACGCTAAACCTGAC2400    AGAAGTGGTGAAAAGGCAGAACTCCAAATCCAAGAAGGGCTTTAACCAGATTAGCACATC2460    GCAGATCAAAGTGGACAAGGTGCAGATCATCGGCTCCAACAGCTGCCCCTTTGCTGTGTG2520    CCTGGACCAGGATGAGAGAAAGATCCTGCAGAGTGTAGTCAGATGTGAGGTCTCACCGTG2580    CTACAAGCCAGAGAAGAGCGACCTCTCCTCACCACCCCAGACGCCTCCTGACCTGCCGGC2640    CCAGGCCGCACCTGATCTCTGCTCCCTTCTCTGCCTGCCCATCATGACTTTCAGTGGAGC2700    TCTGCCCTAGTGTGGGCCCAGCGCCAGACTGGACAGAAGCCCTGGGGCAACCTCCTCGGC2760    CACCCCTCCAGGACAGTCCCTCTCTGTGGAGAACTGAATGGCCCTGTGCAGAGCCATAGT2820    CCCACTGTGGGTCCTGCAATGAGCAGGGGCTGGGAGTAGAGGGTTTCTGGGGCCTCAGGG2880    TTCTGGGAAAGCAACAGCTATCAGAGAGAGAAGGGCCAGACCCCATAGCCTCTTAGATTC2940    CTGGCAGTAGAAGGAGAAGGATGGGTAAATTGACCTCTGAAGTCCCTGACCATTAGCATG3000    GTCTAGGATCCTTTCTAGAAGGAAGATCTGAGGCTCTGGTGCTCAGGGGGATGGCTTGGG3060    CCTTTTCTCTCAACCTTGGCTGAGCCTACCCCTTACTTTGCCAAAGACTTGAGGACCCTG3120    TATGTCTGGAGTTCAGTCCCCTCCTCTGTGGGGCTCAGGTGATTGAAATGTGGATGAAAC3180    ATTTCTCTACTTCAAGACCACCTCTCCCTGCAAACACCACACACACATGGCATGCATGTA3240    CGCACATGCGCACCG3255    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 880 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    MetGlnProGlyAlaThrThrCysThrGluAspArgIleGlnHisAla    151015    LeuGluArgCysLeuHisGlyLeuSerLeuSerArgArgSerThrSer    202530    TrpSerAlaGlyLeuCysLeuAsnCysTrpSerLeuGlnGluLeuVal    354045    SerArgAspProGlyHisPheLeuIleLeuLeuGluGlnIleLeuGln    505560    LysThrArgGluValGlnGluLysGlyThrTyrAspLeuLeuThrPro    65707580    LeuAlaLeuLeuPheTyrSerThrValLeuCysThrProHisPhePro    859095    ProAspSerAspLeuLeuLeuLysAlaAlaSerThrTyrHisArgPhe    100105110    LeuThrTrpProValProTyrCysSerIleCysGlnGluLeuLeuThr    115120125    PheIleAspAlaGluLeuLysAlaProGlyIleSerTyrGlnArgLeu    130135140    ValArgAlaGluGlnGlyLeuProIleArgSerHisArgSerSerThr    145150155160    ValThrValLeuLeuLeuAsnProValGluValGlnAlaGluPheLeu    165170175    AlaValAlaAsnLysLeuSerThrProGlyHisSerProHisSerAla    180185190    TyrThrThrLeuLeuLeuHisAlaPheGlnAlaThrPheGlyAlaHis    195200205    CysAspValProGlyLeuHisCysArgLeuGlnAlaLysThrLeuAla    210215220    GluLeuGluAspIlePheThrGluThrAlaGluAlaGlnGluLeuAla    225230235240    SerGlyIleGlyAspAlaAlaGluAlaArgArgTrpLeuArgThrLys    245250255    LeuGlnAlaValGlyGluLysAlaGlyPheProGlyValLeuAspThr    260265270    AlaLysProGlyLysLeuHisThrIleProIleProValAlaArgCys    275280285    TyrThrTyrSerTrpSerGlnAspSerPheAspIleLeuGlnGluIle    290295300    LeuLeuLysGluGlnGluLeuLeuGlnProGlyIleLeuGlyAspAsp    305310315320    GluGluGluGluGluGluGluGluGluValGluGluAspLeuGluThr    325330335    AspGlyHisCysAlaGluArgAspSerLeuLeuSerThrSerSerLeu    340345350    AlaSerHisAspSerThrLeuSerLeuAlaSerSerGlnAlaSerGly    355360365    ProAlaLeuSerArgHisLeuLeuThrSerPheValSerGlyLeuSer    370375380    AspGlyMetAspSerGlyTyrValGluAspSerGluGluSerSerSer    385390395400    GluTrpProTrpArgArgGlySerGlnGluArgArgGlyHisArgArg    405410415    ProGlyGlnLysPheIleArgIleTyrLysLeuPheLysSerThrSer    420425430    GlnLeuValLeuArgArgAspSerArgSerLeuGluGlySerSerAsp    435440445    ThrAlaLeuProLeuArgArgAlaGlySerLeuCysSerProLeuAsp    450455460    GluProValSerProProSerArgAlaGlnArgSerArgSerLeuPro    465470475480    GlnProLysLeuGlyThrGlnLeuProSerTrpLeuLeuAlaProAla    485490495    SerArgProGlnArgArgArgProPheLeuSerGlyAspGluAspPro    500505510    LysAlaSerThrLeuArgValValValPheGlySerAspArgIleSer    515520525    GlyLysValAlaArgAlaTyrSerAsnLeuArgArgLeuGluAsnAsn    530535540    ArgProLeuLeuThrArgPhePheLysLeuGlnPhePheTyrValPro    545550555560    ValLysArgSerHisGlyThrSerProGlyAlaCysProProProArg    565570575    SerGlnThrProSerProProThrAspSerProArgHisAlaSerPro    580585590    GlyGluLeuGlyThrThrProTrpGluGluSerThrAsnAspIleSer    595600605    HisTyrLeuGlyMetLeuAspProTrpTyrGluArgAsnValLeuGly    610615620    LeuMetHisLeuProProGluValLeuCysGlnGlnSerLeuLysAla    625630635640    GluAlaGlnAlaLeuGluGlySerProThrGlnLeuProIleLeuAla    645650655    AspMetLeuLeuTyrTyrCysArgPheAlaAlaArgProValLeuLeu    660665670    GlnLeuTyrGlnThrGluLeuThrPheIleThrGlyGluLysThrThr    675680685    GluIlePheIleHisSerLeuGluLeuGlyHisSerAlaAlaThrArg    690695700    AlaIleLysAlaSerGlyProGlySerLysArgLeuGlyIleAspGly    705710715720    AspArgGluAlaValProLeuThrLeuGlnIleIleTyrSerLysGly    725730735    AlaIleSerGlyArgSerArgTrpSerAsnLeuGluLysValCysThr    740745750    SerValAsnLeuAsnLysAlaCysArgLysGlnGluGluLeuAspSer    755760765    SerMetGluAlaLeuThrLeuAsnLeuThrGluValValLysArgGln    770775780    AsnSerLysSerLysLysGlyPheAsnGlnIleSerThrSerGlnIle    785790795800    LysValAspLysValGlnIleIleGlySerAsnSerCysProPheAla    805810815    ValCysLeuAspGlnAspGluArgLysIleLeuGlnSerValValArg    820825830    CysGluValSerProCysTyrLysProGluLysSerAspLeuSerSer    835840845    ProProGlnThrProProAspLeuProAlaGlnAlaAlaProAspLeu    850855860    CysSerLeuLeuCysLeuProIleMetThrPheSerGlyAlaLeuPro    865870875880    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5162 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    TTTGAATTTGTTTTGTTTTCAAAAATTAAACAAATGATCCTTCAGCATCATCGCCTCCGC60    TGCTTTATCAGGTCGCATAGGGCATGGAGCTGGAGAACTATAAACAGCCCGTGGTGCTGA120    GAGAGGACAACTGCCGAAGGCGCCGGAGGATGAAGCCGCGCAGTGCTGCGGCCAGCCTGT180    CCTCCATGGAGCTCATCCCCATCGAGTTCGTGCTGCCCACCAGCCAGCGCAAATGCAAGA240    GCCCCGAAACGGCGCTGCTGCACGTGGCCGGCCACGGCAACGTGGAGCAGATGAAGGCCC300    AGGTGTGGCTGCGAGCGCTGGAGACCAGCGTGGCGGCGGACTTCTACCACCGGCTGGGAC360    CGCATCACTTCCTCCTGCTCTATCAGAAGAAGGGGCAGTGGTACGAGATCTACGACAAGT420    ACCAGGTGGTGCAGACTCTGGACTGCCTGCGCTACTGGAAGGCCACGCACCGGAGCCCGG480    GCCAGATCCACCTGGTGCAGCGGCACCCGCCCTCCGAGGAGTCCCAAGCCTTCCAGCGGC540    AGCTCACGGCGCTGATTGGCTATGACGTCACTGACGTCAGCAACGTGCACGACGATGAGC600    TGGAGTTCACGCGCCGTGGCTTGGTGACCCCGCGCATGGCGGAGGTGGCCAGCCGCGACC660    CCAAGCTCTACGCCATGCACCCGTGGGTGACGTCCAAGCCCCTCCCGGAGTACCTGTGGA720    AGAAGATTGCCAACAACTGCATCTTCATCGTCATTCACCGCAGCACCACCAGCCAGACCA780    TTAAGGTCTCACCCGACGACACCCCCGGCGCCATCCTGCAGAGCTTCTTCACCAAGATGG840    CCAAGAAGAAATCTCTGATGGATATTCCCGAAAGCCAAAGCGAACAGGATTTTGTGCTGC900    GCGTCTGTGGCCGGGATGAGTACCTGGTGGGCGAAACGCCCATCAAAAACTTCCAGTGGG960    TGAGGCACTGCCTCAAGAACGGAGAAGAGATTCACGTGGTACTGGACACGCCTCCAGACC1020    CGGCCCTAGACGAGGTGAGGAAGGAAGAGTGGCCGCTGGTGGACGACTGCACGGGAGTCA1080    CCGGCTACCATGAGCAGCTTACCATCCACGGCAAGGACCACGAGAGTGTGTTCACCGTGT1140    CCCTGTGGGACTGCGACCGCAAGTTCAGGGTCAAGATCAGAGGCATTGATATCCCCGTCC1200    TGCCTCGGAACACCGACCTCACAGTTTTTGTAGAGGCAAACATCCAGCATGGGCAACAAG1260    TCCTTTGCCAAAGGAGAACCAGCCCCAAACCCTTCACAGAGGAGGTGCTGTGGAATGTGT1320    GGCTTGAGTTCAGTATCAAAATCAAAGACTTGCCCAAAGGGGCTCTACTGAACCTCCAGA1380    TCTACTGCGGTAAAGCTCCAGCACTGTCCAGCAAGGCCTCTGCAGAGTCCCCCAGTTCTG1440    AGTCCAAGGGCAAAGTTCGGCTTCTCTATTATGTGAACCTGCTGCTGATAGACCACCGTT1500    TCCTCCTGCGCCGTGGAGAATACGTCCTCCACATGTGGCAGATATCTGGGAAGGGAGAAG1560    ACCAAGGAAGCTTCAATGCTGACAAACTCACGTCTGCAACTAACCCAGACAAGGAGAACT1620    CAATGTCCATCTCCATTCTTCTGGACAATTACTGCCACCCGATAGCCCTGCCTAAGCATC1680    AGCCCACCCCTGACCCGGAAGGGGACCGGGTTCGAGCAGAAATGCCCAACCAGCTTCGCA1740    AGCAATTGGAGGCGATCATAGCCACTGATCCACTTAACCCTCTCACAGCAGAGGACAAAG1800    AATTGCTCTGGCATTTTAGATACGAAAGCCTTAAGCACCCAAAAGCATATCCTAAGCTAT1860    TTAGTTCAGTGAAATGGGGACAGCAAGAAATTGTGGCCAAAACATACCAATTGTTGGCCA1920    GAAGGGAAGTCTGGGATCAAAGTGCTTTGGATGTTGGGTTAACAATGCAGCTCCTGGACT1980    GCAACTTCTCAGATGAAAATGTAAGAGCCATTGCAGTTCAGAAACTGGAGAGCTTGGAGG2040    ACGATGATGTTCTGCATTACCTTCTACAATTGGTCCAGGCTGTGAAATTTGAACCATACC2100    ATGATAGCGCCCTTGCCAGATTTCTGCTGAAGCGTGGTTTAAGAAACAAAAGAATTGGTC2160    ACTTTTTGTTTTGGTTCTTGAGAAGTGAGATAGCCCAGTCCAGACACTATCAGCAGAGGT2220    TCGCTGTGATTCTGGAAGCCTATCTGAGGGGCTGTGGCACAGCCATGCTGCACGACTTTA2280    CCCAACAAGTCCAAGTAATCGAGATGTTACAAAAAGTCACCCTTGATATTAAATCGCTCT2340    CTGCTGAAAAGTATGACGTCAGTTCCCAAGTTATTTCACAACTTAAACAAAAGCTTGAAA2400    ACCTGCAGAATTCTCAACTCCCCGAAAGCTTTAGAGTTCCATATGATCCTGGACTGAAAG2460    CAGGAGCGCTGGCAATTGAAAAATGTAAAGTAATGGCCTCCAAGAAAAAACCACTATGGC2520    TTGAGTTTAAATGTGCCGATCCTACAGCCCTATCAAATGAAACAATTGGAATTATCTTTA2580    AACATGGTGATGATCTGCGCCAAGACATGCTTATTTTACAGATTCTACGAATCATGGAGT2640    CTATTTGGGAGACTGAATCTTTGGATCTATGCCTCCTGCCATATGGTTGCATTTCAACTG2700    GTGACAAAATAGGAATGATCGAGATTGTGAAAGACGCCACGACAATTGCCAAAATTCAGC2760    AAAGCACAGTGGGCAACACGGGAGCATTTAAAGATGAAGTCCTGAATCACTGGCTCAAAG2820    AAAAATCCCCTACTGAAGAAAAGTTTCAGGCAGCAGTGGAGAGATTTGTTTATTCCTGTG2880    CAGGCTACTGTGTGGCAACCTTTGTTCTTGGAATAGGCGACAGACACAATGACAATATTA2940    TGATCACCGAGACAGGAAACCTATTTCATATTGACTTCGGGCACATTCTTGGGAATTACA3000    AAAGTTTCCTGGGCATTAATAAAGAGAGAGTGCCATTTGTGCTAACCCCTGACTTCCTCT3060    TTGTGATGGGAACTTCTGGAAAGAAGACAAGCCCACACTTCCAGAAATTTCAGGACATCT3120    GTGTTAAGGCTTATCTAGCCCTTCGTCATCACACAAACCTACTGATCATCCTGTTCTCCA3180    TGATGCTGATGACAGGAATGCCCCAGTTAACAAGCAAAGAAGACATTGAATATATCCGGG3240    ATGCCCTCACAGTGGGGAAAAATGAGGAGGATGCTAAAAAGTATTTTCTTGATCAGATCG3300    AAGTTTGCAGAGACAAAGGATGGACTGTGCAGTTTAATTGGTTTCTACATCTTGTTCTTG3360    GCATCAAACAAGGAGAGAAACATTCAGCCTAATACTTTAGGCTAGAATCAAAAACAAGTT3420    AGTGTTCTATGGTTTAAATTAGCATAGCAATCATCGAACTTGGATTTCAAATGCAATAGA3480    CATTGTGAAAGCTGGCATTTCAGAAGTATAGCTCTTTTCCTACCTGAACTCTTCCCTGGA3540    GAAAAGATGTTGGCATTGCTGATTGTTTGGTTAAGCAATGTCCAGTGCTAGGATTATTTG3600    CAGGTTTGGTTTTTTCTCATTTGTCTGTGGCATTGGAGAATATTCTTGGTTTAAACAGAC3660    TAATGACTTCCTTATTGTCCCTGATATTTTGACTATCTTACTATTGAGTGCTTCTGGAAA3720    TTCTTTGGAATAATTGATGACATCTATTTTCATCTGGGTTTAGTCTCAATTTTGGTTATC3780    TTTGTGTTCCTCAAGCTCTTTAAAGAAAAAGATGTAATCGTTGTAACCTTTGTCTCATTC3840    CTTAAATGATGCTTCCAAACATCTCCTTAGTGTCTGCAGGTGTTAGTGGTGTGCTAAAAG3900    CAAGGAAAGCGAGTTAGTCTTTTCAGTGTCTTTTGCAATTCAATTCTTTTGTCATGTATA3960    ACTGAGACACACAAACACAGCAGGAGAAATCTAAACCGTTGTGCCTTGACCTTCCTCTGC4020    TGGTCTTGTTCCAGGGTTATGAATATGAAAAAATAGAGATGAGACTTTTTGTGTCAACTC4080    TGTCCACAAGAGTGAGTTATCTAGTATGATTAGTATAGCTTTCTCCAGCATGGCAGCAGG4140    AAGTAACTACAGGGCCTCTTTTATGCCTGACATTTCTTCCCTTCCTTTTTCCCTGCCTCC4200    CTTTTTCATCAATTGCAATGCTCCCACAACTCTTTACAGACTTGTGAAATCTTCAAGAAC4260    ACCTTTACTCTATAACTCAAAAATTAGTTGAAAAATAATTACTTCTCAAGGATTATTAGA4320    ATCTTAGGTACTTATTTGTAAAGATGTTTAGTGACTTTTTTTTCAAGTATCTATAAAGGA4380    GGCAGATTCTAGAAAATATGAATTAGTTTCCAAATGCCTTAATTTTAAACTTTGGCCTGA4440    ACAGTTTTTTCTTTTTCTTAATGGAAGAAGATATTTAATATCTTAAAAATATTCCAAGTT4500    AGGAAGAACACTACTTGCCTTATCCATTTCCCATTTAAAGGACTTTTAAACTTTGACACA4560    GTCCTTCAGATTTCCTGAAAATCCTTGAAATATCTTACTTTAAAAATATTTTCATCTCTG4620    AAATATCTCGTTATTTATTGGAGGTATTGTTTAACCTTAGATAGACCATTAAATTATTTA4680    TAAAATATTTTGTAATTACCTGTAGYTAATACATTACATAGAAAAAACTATGTTAACAGT4740    GTCTCTGTTTAAGTATAATCAGATATAAATATATAACTTAATTTTTTAATTTTAAAAATA4800    GATACCTGTTTGACTTTGAGGTAGTCCAGACCTTTTCTTTTTTTTTTTTTTTTTTAATGT4860    GTGCAAAAGCCCAAAGGTTCCTAAGCCTGGCTGCAAAGAAGAATCAACAGGGACACTTTT4920    TAAAAACACTCTTATCAGCCTGGGCAACACAGTGAGACTCCATCTCTTAAAAAAAAAATT4980    AGCTGGGTATAGTGGTATGTGCCTGTAGTCCCAGGTACTCAGGAGGCTGASGCAGGAGGA5040    TTGCCTGAGCCCAGGAGGTGGAAACTGCAGAGAGTCATGATCATGTCCTTACACTCCAGC5100    CTGGATAACAGAGCGAGACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAGTCGACCG5160    AG5162    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1101 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: None    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    MetGluLeuGluAsnTyrLysGlnProValValLeuArgGluAspAsn    151015    CysArgArgArgArgArgMetLysProArgSerAlaAlaSerLeuSer    202530    SerMetGluLeuIleProIleGluPheValLeuProThrSerGlnArg    354045    LysCysLysSerProGluThrAlaLeuLeuHisValAlaGlyHisGly    505560    AsnValGluGlnMetLysAlaGlnValTrpLeuArgAlaLeuGluThr    65707580    SerValAlaAlaAspPheTyrHisArgLeuGlyProHisHisPheLeu    859095    LeuLeuTyrGlnLysLysGlyGlnTrpTyrGluIleTyrAspLysTyr    100105110    GlnValValGlnThrLeuAspCysLeuArgTyrTrpLysAlaThrHis    115120125    ArgSerProGlyGlnIleHisLeuValGlnArgHisProProSerGlu    130135140    GluSerGlnAlaPheGlnArgGlnLeuThrAlaLeuIleGlyTyrAsp    145150155160    ValThrAspValSerAsnValHisAspAspGluLeuGluPheThrArg    165170175    ArgGlyLeuValThrProArgMetAlaGluValAlaSerArgAspPro    180185190    LysLeuTyrAlaMetHisProTrpValThrSerLysProLeuProGlu    195200205    TyrLeuTrpLysLysIleAlaAsnAsnCysIlePheIleValIleHis    210215220    ArgSerThrThrSerGlnThrIleLysValSerProAspAspThrPro    225230235240    GlyAlaIleLeuGlnSerPhePheThrLysMetAlaLysLysLysSer    245250255    LeuMetAspIleProGluSerGlnSerGluGlnAspPheValLeuArg    260265270    ValCysGlyArgAspGluTyrLeuValGlyGluThrProIleLysAsn    275280285    PheGlnTrpValArgHisCysLeuLysAsnGlyGluGluIleHisVal    290295300    ValLeuAspThrProProAspProAlaLeuAspGluValArgLysGlu    305310315320    GluTrpProLeuValAspAspCysThrGlyValThrGlyTyrHisGlu    325330335    GlnLeuThrIleHisGlyLysAspHisGluSerValPheThrValSer    340345350    LeuTrpAspCysAspArgLysPheArgValLysIleArgGlyIleAsp    355360365    IleProValLeuProArgAsnThrAspLeuThrValPheValGluAla    370375380    AsnIleGlnHisGlyGlnGlnValLeuCysGlnArgArgThrSerPro    385390395400    LysProPheThrGluGluValLeuTrpAsnValTrpLeuGluPheSer    405410415    IleLysIleLysAspLeuProLysGlyAlaLeuLeuAsnLeuGlnIle    420425430    TyrCysGlyLysAlaProAlaLeuSerSerLysAlaSerAlaGluSer    435440445    ProSerSerGluSerLysGlyLysValArgLeuLeuTyrTyrValAsn    450455460    LeuLeuLeuIleAspHisArgPheLeuLeuArgArgGlyGluTyrVal    465470475480    LeuHisMetTrpGlnIleSerGlyLysGlyGluAspGlnGlySerPhe    485490495    AsnAlaAspLysLeuThrSerAlaThrAsnProAspLysGluAsnSer    500505510    MetSerIleSerIleLeuLeuAspAsnTyrCysHisProIleAlaLeu    515520525    ProLysHisGlnProThrProAspProGluGlyAspArgValArgAla    530535540    GluMetProAsnGlnLeuArgLysGlnLeuGluAlaIleIleAlaThr    545550555560    AspProLeuAsnProLeuThrAlaGluAspLysGluLeuLeuTrpHis    565570575    PheArgTyrGluSerLeuLysHisProLysAlaTyrProLysLeuPhe    580585590    SerSerValLysTrpGlyGlnGlnGluIleValAlaLysThrTyrGln    595600605    LeuLeuAlaArgArgGluValTrpAspGlnSerAlaLeuAspValGly    610615620    LeuThrMetGlnLeuLeuAspCysAsnPheSerAspGluAsnValArg    625630635640    AlaIleAlaValGlnLysLeuGluSerLeuGluAspAspAspValLeu    645650655    HisTyrLeuLeuGlnLeuValGlnAlaValLysPheGluProTyrHis    660665670    AspSerAlaLeuAlaArgPheLeuLeuLysArgGlyLeuArgAsnLys    675680685    ArgIleGlyHisPheLeuPheTrpPheLeuArgSerGluIleAlaGln    690695700    SerArgHisTyrGlnGlnArgPheAlaValIleLeuGluAlaTyrLeu    705710715720    ArgGlyCysGlyThrAlaMetLeuHisAspPheThrGlnGlnValGln    725730735    ValIleGluMetLeuGlnLysValThrLeuAspIleLysSerLeuSer    740745750    AlaGluLysTyrAspValSerSerGlnValIleSerGlnLeuLysGln    755760765    LysLeuGluAsnLeuGlnAsnSerGlnLeuProGluSerPheArgVal    770775780    ProTyrAspProGlyLeuLysAlaGlyAlaLeuAlaIleGluLysCys    785790795800    LysValMetAlaSerLysLysLysProLeuTrpLeuGluPheLysCys    805810815    AlaAspProThrAlaLeuSerAsnGluThrIleGlyIleIlePheLys    820825830    HisGlyAspAspLeuArgGlnAspMetLeuIleLeuGlnIleLeuArg    835840845    IleMetGluSerIleTrpGluThrGluSerLeuAspLeuCysLeuLeu    850855860    ProTyrGlyCysIleSerThrGlyAspLysIleGlyMetIleGluIle    865870875880    ValLysAspAlaThrThrIleAlaLysIleGlnGlnSerThrValGly    885890895    AsnThrGlyAlaPheLysAspGluValLeuAsnHisTrpLeuLysGlu    900905910    LysSerProThrGluGluLysPheGlnAlaAlaValGluArgPheVal    915920925    TyrSerCysAlaGlyTyrCysValAlaThrPheValLeuGlyIleGly    930935940    AspArgHisAsnAspAsnIleMetIleThrGluThrGlyAsnLeuPhe    945950955960    HisIleAspPheGlyHisIleLeuGlyAsnTyrLysSerPheLeuGly    965970975    IleAsnLysGluArgValProPheValLeuThrProAspPheLeuPhe    980985990    ValMetGlyThrSerGlyLysLysThrSerProHisPheGlnLysPhe    99510001005    GlnAspIleCysValLysAlaTyrLeuAlaLeuArgHisHisThrAsn    101010151020    LeuLeuIleIleLeuPheSerMetMetLeuMetThrGlyMetProGln    1025103010351040    LeuThrSerLysGluAspIleGluTyrIleArgAspAlaLeuThrVal    104510501055    GlyLysAsnGluGluAspAlaLysLysTyrPheLeuAspGlnIleGlu    106010651070    ValCysArgAspLysGlyTrpThrValGlnPheAsnTrpPheLeuHis    107510801085    LeuValLeuGlyIleLysGlnGlyGluLysHisSerAla    109010951100    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule which encodes ap101 regulatory subunit having a nucleotide sequence that:a) encodes SEQID NO:12; or b) hybridizes under highly stringent conditions to thenucleotide sequence of (a) or to its complement.
 2. The isolated nucleicacid molecule according to claim 1, wherein the nucleic acid moleculehybridizes under highly stringent conditions to the nucleotide sequenceof (a) or to its complement.
 3. The isolated nucleic acid moleculeaccording to claim 1, wherein the nucleic acid molecule encodes SEQ IDNO:12.
 4. An isolated nucleotide sequence encoding a polypeptidecorresponding to the catalytic subunit interaction domain, or the Gβγinteraction domain, of the p101 regulatory subunit of claim 1; or adeletion mutant of the p101 regulatory subunit of claim 1, in which thecatalytic subunit interaction domain, or the Gβγ interaction domain, isdeleted.
 5. An isolated nucleotide sequence encoding a chimeric proteincomprising the polypeptide encoded by claim 4, fused to a heterologouspolypeptide.
 6. The isolated nucleotide sequence of claim 5 in which theheterologous polypeptide is a Glu tag or a myc epitope tag.
 7. Anucleotide vector containing the nucleotide sequence of claim
 1. 8. Anexpression vector containing the nucleotide sequence of claim 1 inoperative association with a nucleotide regulatory sequence thatcontrols expression of the nucleotide sequence in a host cell.
 9. Theexpression vector of claim 8, in which said regulatory sequence isselected from the group consisting of the cytomegalovirus hCMV immediateearly gene promoter, the early or late promoters of SV40 adenovirus, thebaculovirus promoter, the lac system, the trp system, the TAC system,the TRC system, the major operator and promoter regions of phage λ, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase, the promoters of acid phosphatase, and the promoters of theyeast α-mating factors.
 10. A host cell that has been geneticallyengineered to contain the nucleotide sequence of claim
 1. 11. The hostcell according to claim 10, wherein the host cell has been geneticallyengineered to contain the nucleotide sequence of claim 1 in operativeassociation with a nucleotide regulatory sequence that controlsexpression of the nucleotide sequence in the host cell.
 12. The hostcell of claim 11 in which the host cell is a Sf9 cell, a Chinese hamsterovary cell, a COS cell, or a U937 cell.
 13. An isolated nucleic acidmolecule which encodes a p120 catalytic subunit having a nucleotidesequence that:a) encodes SEQ ID NO:12; or b) hybridizes under highlystringent conditions to at least the nucleotide sequence of (a) encodingthe last 28 amino acids of the p120 protein, or to its complement. 14.The isolated nucleic acid molecule according to claim 13, wherein thenucleic acid molecule hybridizes under highly stringent conditions to atleast the nucleotide sequence of SEQ ID NO:13 that encodes the last 28amino acids of the p120 protein, or to its complement.
 15. The isolatednucleic acid molecule according to claim 14, wherein the nucleic acidmolecule encodes SEQ ID NO:14.
 16. An isolated nucleotide sequenceencoding a chimeric protein comprising the p120 regulatory subunit ofclaim 13 fused to a heterologous polypeptide.
 17. The isolatednucleotide sequence of claim 16 in which the heterologous polypeptide isa Glu tag or a myc epitope tag.
 18. A nucleotide vector containing thenucleotide sequence of claim
 13. 19. An expression vector containing thenucleotide sequence of claim 13 in operative association with anucleotide regulatory sequence that controls expression of thenucleotide sequence in a host cell.
 20. The expression vector of claim19, in which said regulatory sequence is selected from the groupconsisting of the cytomegalovirus hCMV immediate early gene promoter,the early or late promoters of SV40 adenovirus, the baculoviruspromoter, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage λ, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors.
 21. A host cell that has been genetically engineeredto contain the nucleotide sequence of claim
 13. 22. The host cellaccording to claim 21, wherein the host cell has been geneticallyengineered to contain the nucleotide sequence of claim 14 in operativeassociation with a nucleotide regulatory sequence that controlsexpression of the nucleotide sequence in the host cell.
 23. The hostcell of claim 22 in which the host cell is a Sf9 cell, a Chinese hamsterovary cell, a COS cell, or a U937 cell.
 24. The host cell according toclaim 10, wherein the host cell has been genetically engineered tocontain the nucleotide sequence of claim
 13. 25. An isolated nucleicacid molecule which encodes a p101 regulatory subunit, or a fragmentthereof, comprising a nucleotide sequence that encodes an amino acidsequence selected from the group consisting of amino acid residues 1-160of SEQ ID NO:12, amino acid residues 161-263 of SEQ ID NO:12, amino acidresidues 1-735 of SEQ ID NO:12; and amino acid residues 736-880 of SEQID NO:12.
 26. An isolated nucleic acid molecule comprising a nucleotidesequence that encodes the amino acid sequence corresponding to residue41 to residue 1101 of SEQ ID NO:14.
 27. An isolated nucleic acidmolecule comprising a nucleotide sequence that encodes amino acidresidues 173-302 of SEQ ID NO:14.
 28. An isolated nucleic acid moleculeencoding a p101 regulatory subunit and having a nucleotide sequence thathybridizes under highly stringent conditions to the nucleotide sequenceshown in SEQ ID NO:11, or its complement.
 29. The isolated nucleic acidmolecule of claim 28 having the nucleotide sequence shown in SEQ IDNO:11.
 30. A polynucleotide encoding a chimeric protein comprising thep101 regulatory subunit of claim 28 fused to a heterologous polypeptide.31. A nucleotide vector containing the nucleotide sequence of claim 28.32. A host cell that has been genetically engineered to contain thenucleotide sequence of claim 28.